CN112481283A

CN112481283A - Treatment of cancer using CD33 chimeric antigen receptor

Info

Publication number: CN112481283A
Application number: CN202011387472.9A
Authority: CN
Inventors: J·布罗格顿; H·艾伯斯巴赫; S·吉尔; D·格拉斯; T·胡贝尔; J·雅斯库; S·肯德利安; J·曼尼克; M·C·米伦; L·墨菲; C·理查森; R·辛格; 宋慧娟; 吴期隆; 张继全
Original assignee: Novartis AG; University of Pennsylvania Penn
Current assignee: Novartis AG; University of Pennsylvania Penn
Priority date: 2014-07-21
Filing date: 2015-07-21
Publication date: 2021-03-12
Also published as: RU2017105160A; US10851166B2; JP6831777B2; US9777061B2; BR112017001242A2; SG10201913765YA; US20160096892A1; US20180044423A1; CO2017000510A2; ZA201700157B; KR102594343B1; MX2017001008A; KR20170029009A; AU2015292755A1; IL250138A0; CA2955154A1; CN107109419B; CN107109419A; EP3172234B1; WO2016014576A1

Abstract

The present disclosure provides compositions and methods for treating diseases associated with expression of CD 33. The disclosure also relates to Chimeric Antigen Receptors (CARs) specific for CD33, vectors encoding the same, and recombinant T cells comprising CD33 CARs. The disclosure also includes methods of administering a genetically modified T cell expressing a CAR comprising a CD33 binding domain.

Description

Treatment of cancer using CD33 chimeric antigen receptor

The application is a divisional application of Chinese patent application 201580050572.3, the application date of the original application is 21/7/2015, and the name of the invention is 'treating cancer by using CD33 chimeric antigen receptor'.

This application claims priority to PCT application number PCT/CN2014/082589, day 7/21 2014 and PCT application number PCT/CN2014/090504, day 11/6 2014. The entire contents of these applications are incorporated herein by reference.

Sequence listing

This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on day 15/7/2015 was named N2067-7047WO3_ sl. txt and was 323,686 bits in size.

Technical Field

The present invention relates generally to the use of immune effector cells (e.g., T cells, NK cells) engineered to express a Chimeric Antigen Receptor (CAR) for treating diseases associated with expression of the differentiation antigen cluster 33 protein (CD 33).

Background

Most Acute Myeloid Leukemia (AML) patients are incurable using standard therapy (Mrozek et al, 2012, J Clin Oncol,30:4515-23) and those with relapsed or refractory AML (RR-AML) have a particularly poor prognosis (Kern et al, 2003, Blood 2003,101: 64-70; Wheatley et al, 1999, Br J Haematol,107: 69-79).

Genetic engineering can confer specificity to T cells for the chosen target. T cells can be transduced with genetic material encoding an antibody single chain variable fragment (scFv) along with signaling molecules, thus utilizing Complementarity Determining Regions (CDRs) to recognize cell surface antigens in a non-MHC-restricted manner. These cells are called Chimeric Antigen Receptor (CAR) T cells. Preclinical and clinical attempts to target at least 20 different surface molecules in a variety of malignancies have shown some activity, however these attempts are often limited by poor persistence of the infused CAR T cell product (Sadelain et al, 2009, Curr Opin Immunol 2009,21: 215-23). Recent success when using anti-CD 19 redirecting T cells in patients with advanced Chronic Lymphoid Leukemia (CLL) and Acute Lymphoid Leukemia (ALL) (Porter et al 2011, N Engl J Med,365: 725-33; Kalos et al 2011, Science trans Med,3:95ra 73; Grupp and Kalos,2013, N Engl J Med,368:1509-18) showed that these cells could eradicate a large tumor burden after a single infusion, with remission lasting up to now for 3 years, thus emphasizing the great potential of CAR T cell therapy. There have been few preclinical attempts to target AML in animal models (Marin et al, 2010, haematalogica, 95: 2144-52; tettamani et al, 2013, Br J Haematol,161: 389-401). A recently published small clinical trial showed that it was feasible to generate T cells and infuse them into aggressive malignant patients (Ritchie et al, 2013, Mol Ther,2013 Nov; 21(11): 2122-9). In addition to the ability of chimeric antigen receptors on genetically modified T cells to recognize and destroy targeted cells, successful therapeutic T cell therapy needs to have the ability to proliferate and persist over time and further monitor leukemia cell escape. Variable T cell quality, whether this is due to disability, suppression or failure, can affect the expression of CAR-transformed T cells. The skilled practitioner now has limited control over the variability of T cell mass. To be effective, CAR-transformed patient T cells need to persist and maintain the ability to proliferate in response to the antigen of the CAR. It has been shown that T cells from ALL patients can do this with CART19 containing murine scFv (see, e.g., Grupp et al, NEJM 368:1509-1518 (2013)).

Summary of The Invention

In a first aspect, the invention features an isolated nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises an antibody or antibody fragment comprising a CD33 binding domain (e.g., a human or humanized CD33 binding domain), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain). In one embodiment, the CAR comprises an antibody or antibody fragment comprising a CD33 binding domain described herein (e.g., a human or humanized CD33 binding domain described herein), a transmembrane domain described herein, and an intracellular signaling domain described herein (e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain).

In one embodiment, the encoded CD33 binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1(LC CDR1), light chain complementarity determining region 2(LC CDR2), and light chain complementarity determining region 3(LC CDR3) of the CD33 binding domain described herein, and/or one or more (e.g., all three) heavy chain complementarity determining region 1(HC CDR1), heavy chain complementarity determining region 2(HC CDR2), and heavy chain complementarity determining region 3(HC CDR3) of the CD33 binding domain described herein, e.g., a CD33 binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs. In one embodiment, the encoded CD33 binding domain (e.g., a human or humanized CD33 binding domain) comprises a light chain variable region described herein (e.g., in table 2 or table 9) and/or a heavy chain variable region described herein (e.g., in table 2 or table 9). In one embodiment, the encoded CD33 binding domain is an scFv comprising a light chain and a heavy chain of the amino acid sequences of table 2 or table 9. In one embodiment, the encoded CD33 binding domain (e.g., scFv) comprises a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of the light chain variable region provided in table 2 or 9, but no more than 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence having 95-99% identity to the amino acid sequence of table 2 or 9; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of the heavy chain variable region provided in table 2 or 9, but no more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence having 95-99% identity to the amino acid sequence of table 2 or 9.

In other embodiments, the encoded CD33 binding domain comprises the HC CDR1, HC CDR2, and HC CDR3 of any of the CD33 heavy chain binding domain amino acid sequences listed in table 2 or table 9. In embodiments, the CD33 binding domain further comprises LC CDR1, LC CDR2, and LC CDR 3. In embodiments, the CD33 binding domain comprises LC CDR1, LC CDR2, and LC CDR3 of any of the CD33 light chain binding domain amino acid sequences listed in table 2 or table 9.

In some embodiments, the encoded CD33 binding domain comprises one, two, or all of LC CDR1, LC CDR2, and LC CDR3 of any CD33 light chain binding domain amino acid sequence listed in table 2 or table 9 and one, two, or all of HC CDR1, HC CDR2, and HC CDR3 of any CD33 heavy chain binding domain amino acid sequence listed in table 2 or table 9.

In one embodiment, the encoded CD33 binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 39-47, 57-65, 66-74, or 262-268. In one embodiment, the encoded CD33 binding domain (e.g., scFv) comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions, e.g., conservative substitutions) but NO more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of SEQ ID NOS 39-47, 57-65, 66-74 or 262-268 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NOS 39-47, 57-65, 66-74 or 262-268. In another embodiment, the encoded CD33 binding domain comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS 57-65 or a sequence 95-99% identical thereto. In another embodiment, the encoded CD33 binding domain comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS 66-74 or a sequence 95-99% identical thereto. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO 255-261 or a sequence having 95-99% identity thereto.

In one embodiment, the encoded CD33 binding domain comprises a (Gly4-Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4(SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can be, for example, in any of the following orientations: a light chain variable region-linker-heavy chain variable region or a heavy chain variable region-linker-light chain variable region.

In one embodiment, the encoded CAR comprises a transmembrane domain comprising a transmembrane domain of a protein (e.g., a protein described herein, e.g., a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD 154). In one embodiment, the encoded transmembrane domain comprises the sequence of SEQ ID NO 6. In one embodiment, the encoded transmembrane domain comprises an amino acid sequence comprising at least one, two or three modifications, but NO more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO. 6 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 6. In one embodiment, the nucleic acid sequence encoding the transmembrane domain comprises the sequence of SEQ ID NO 17 or a sequence 95-99% identical thereto.

In one embodiment, the encoded CD33 binding domain is linked to the transmembrane domain by a hinge region (e.g., a hinge region as described herein). In one embodiment, the encoded hinge region comprises SEQ ID NO 2 or a sequence 95-99% identical thereto. In one embodiment, the nucleic acid sequence encoding the hinge region comprises the sequence of SEQ ID NO. 13 or a sequence 95-99% identical thereto.

In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a co-stimulatory domain (e.g., a co-stimulatory domain described herein). In embodiments, the intracellular signaling domain comprises a costimulatory domain. In embodiments, the intracellular signaling domain comprises a primary signaling domain. In embodiments, the intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

In one embodiment, the encoded co-stimulatory domain is a functional signal domain obtained from, e.g., a protein as described herein, e.g., a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signal molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1(CD11a/CD a), 4-1BB (CD137), B a-H a, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHT TR), KIRDS a, SLAMF a, NKp a (KLRF a), NKp a, CD a alpha, CD a beta, IL2 a gamma, ITIL 7 alpha, IT3672, VLITGA 72, VLITGA a, GAITGB 72, GAITGA a, GAITGB 11-a, GAITGA a, GAITGB 11, GAITGA a, GAITGB a, GAIT11, CD a, GAITGA a, GAITGB 72, GAITGA a, CD a, GAITGB 11-a, GAITGB 11, GAITGA a, GAITGB a, CD a, GAITGA a, GAITGB 11-a, GAITCD a, GAITGA a, CD a, GAITGB 11-a, CD a, GAITGB 11, SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds to CD 83. In embodiments, the encoded co-stimulatory domain comprises 4-1BB, CD27, CD28, or ICOS.

In one embodiment, the encoded 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO 7. In one embodiment, the encoded co-stimulatory domain comprises an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO. 7 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 7. In one embodiment, the nucleic acid sequence encoding the co-stimulatory domain comprises the nucleotide sequence of SEQ ID NO. 18 or a sequence 95-99% identical thereto. In another embodiment, the encoded CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 379. In one embodiment, the encoded co-stimulatory domain comprises an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO:379 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 379. In one embodiment, the nucleic acid sequence encoding the CD28 co-stimulatory domain comprises the nucleotide sequence of SEQ ID NO. 380 or a sequence with 95-99% identity thereto. In another embodiment, the encoded CD27 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO 8. In one embodiment, the encoded co-stimulatory domain comprises an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO. 8 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 8. In one embodiment, the nucleic acid sequence encoding the CD27 co-stimulatory domain comprises the nucleotide sequence of SEQ ID NO 19 or a sequence with 95-99% identity thereto. In another embodiment, the encoded ICOS co-stimulatory domain comprises the amino acid sequence of SEQ ID NO 381. In one embodiment, the encoded ICOS co-stimulatory domain comprises an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO. 381 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 381. In one embodiment, the nucleic acid sequence encoding the ICOS co-stimulatory domain comprises the nucleotide sequence of SEQ ID NO:382 or a sequence having 95-99% identity thereto.

In embodiments, the encoded primary signaling domain comprises a functional signaling domain of CD3 ζ. In embodiments, the functional signal domain of CD3 ζ comprises the amino acid sequence of SEQ ID NO:9 (mutant CD3 ζ) or SEQ ID NO:10 (wild type human CD3 ζ) or a sequence having 95-99% identity thereto.

In one embodiment, the encoded intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta. In one embodiment, the encoded 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO. 7 and/or the CD3 ζ amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10. In one embodiment, the intracellular signaling domain comprises an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications, of the amino acid sequence of SEQ ID NO. 7 and/or of the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 7 and/or to the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10. In one embodiment, the encoded intracellular signaling domain comprises the sequence of SEQ ID NO 7 and the sequence of SEQ ID NO 9 or SEQ ID NO 10, wherein the sequences comprising the intracellular signaling domain are expressed in frame and as a single polypeptide chain. In one embodiment, the nucleic acid sequence encoding the 4-1BB intracellular signaling domain comprises the nucleotide sequence of SEQ ID NO. 18 or a sequence 95-99% identical thereto, and/or the nucleotide sequence CD3 ζ of SEQ ID NO. 20 or SEQ ID NO. 21 or a sequence 95-99% identical thereto.

In one embodiment, the encoded intracellular signaling domain comprises a functional signaling domain of CD27 and/or a functional signaling domain of CD3 ζ. In one embodiment, the encoded intracellular signaling domain of CD27 comprises the amino acid sequence of SEQ ID NO 8 and/or the amino acid sequence of CD3 ζ of SEQ ID NO 9 or SEQ ID NO 10. In one embodiment, the intracellular signaling domain comprises an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO. 8 and/or the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 8 and/or the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10. In one embodiment, the encoded intracellular signaling domain comprises the sequence of SEQ ID NO 8 and the sequence of SEQ ID NO 9 or SEQ ID NO 10, wherein the sequences comprising the intracellular signaling domain are expressed in frame and as a single polypeptide chain. In one embodiment, the nucleic acid sequence encoding the intracellular signaling domain of CD27 comprises the nucleotide sequence of SEQ ID NO 19 or a sequence having 95-99% identity thereto, and/or the nucleotide sequence of CD3 ζ of SEQ ID NO 20 or SEQ ID NO 21 or a sequence having 95-99% identity thereto.

In one embodiment, the encoded intracellular signaling domain comprises a functional signaling domain of CD28 and/or a functional signaling domain of CD3 ζ. In one embodiment, the encoded intracellular signaling domain of CD28 comprises the amino acid sequence of SEQ ID NO:379 and/or the amino acid sequence of CD3 ζ of SEQ ID NO:9 or SEQ ID NO: 10. In one embodiment, the intracellular signaling domain comprises a sequence having the amino acid sequence of SEQ ID NO:379 and/or at least one, two or three modifications, but NO more than 20, 10 or 5 modifications, of the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10 or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:379 and/or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO: 10. In one embodiment, the encoded intracellular signaling domain comprises the sequence of SEQ ID NO:379 and the sequence of SEQ ID NO:9 or SEQ ID NO:10, wherein the sequences comprising the intracellular signaling domain are expressed in frame and as a single polypeptide chain. In one embodiment, the nucleic acid sequence encoding the intracellular signaling domain of CD28 comprises the nucleotide sequence of SEQ ID NO:380 or a sequence 95-99% identical thereto, and/or the nucleotide sequence CD3 ζ of SEQ ID NO:20 or SEQ ID NO:21 or a sequence 95-99% identical thereto.

In one embodiment, the encoded intracellular signaling domain comprises a functional signaling domain of ICOS and/or a functional signaling domain of CD3 ζ. In one embodiment, the encoded ICOS intracellular signaling domain comprises the amino acid sequence of SEQ ID NO. 381 and/or the CD3 ζ amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10. In one embodiment, the intracellular signaling domain comprises an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications, of the amino acid sequence of SEQ ID NO. 381 and/or of the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10 or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO. 381 and/or to the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10. In one embodiment, the encoded intracellular signaling domain comprises the sequence of SEQ ID NO:381 and the sequence of SEQ ID NO:9 or SEQ ID NO:10, wherein the sequences comprising the intracellular signaling domain are expressed in frame and as a single polypeptide chain. In one embodiment, the nucleic acid sequence encoding the ICOS intracellular signaling domain comprises the nucleotide sequence of SEQ ID NO:382 or a sequence having 95-99% identity thereto, and/or the CD3 zeta nucleotide sequence of SEQ ID NO:20 or SEQ ID NO:21 or a sequence having 95-99% identity thereto.

In another aspect, the invention relates to an isolated nucleic acid molecule encoding a CAR construct comprising a leader sequence, e.g., a leader sequence described herein, e.g., the amino acid sequence of SEQ ID No. 1; a CD33 binding domain as described herein, e.g., a CD33 binding domain comprising LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 as described herein (e.g., a human or humanized CD33 binding domain described in table 2 or table 9), or a sequence 95-99% identical thereto; the hinge region described herein, for example, the amino acid sequence of SEQ ID NO 2; transmembrane domains as described herein, e.g., having the sequence of SEQ ID NO 6; and an intracellular signaling domain, e.g., an intracellular signaling domain as described herein. In one embodiment, the encoded intracellular signaling domain comprises a costimulatory domain, e.g., a costimulatory domain described herein (e.g., a 4-1BB costimulatory domain having the amino acid sequence of SEQ ID NO:7 or a CD27 costimulatory domain having the amino acid sequence of SEQ ID NO: 8), and/or a primary signaling domain, e.g., a primary signaling domain described herein (e.g., a CD3 zeta stimulatory domain having the sequence of SEQ ID NO:9 or SEQ ID NO: 10). In one embodiment, the isolated nucleic acid molecule encoding the CAR construct comprises a leader sequence encoded by the nucleic acid sequence of SEQ ID No. 1 or a sequence having 95-99% identity thereto.

In one embodiment, an isolated nucleic acid molecule comprises (e.g., consists of) a nucleic acid encoding the CAR amino acid sequence of SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, or SEQ ID NO 56; or an amino acid having one, two or three modifications (e.g., substitutions, such as conservative substitutions) but NO more than 30, 20 or 10 modifications (e.g., substitutions, such as conservative substitutions) of the amino acid sequence of

SEQ ID NO

48, 49, 50, 51, 52, 53, 54, 55 or 56; or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of

SEQ ID NO

48, 49, 50, 51, 52, 53, 54, 55 or 56.

In one embodiment, the isolated nucleic acid molecule comprises (e.g., consists of) the nucleic acid sequence of

SEQ ID NO

75, 76, 77, 78, 79, 80, 81, 82 or 83 or a nucleic acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleic acid sequence of

SEQ ID NO

75, 76, 77, 78, 79, 80, 81, 82 or 83.

In one aspect, the invention relates to an isolated nucleic acid molecule encoding a CD33 binding domain, wherein the CD33 binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1(LC CDR1), light chain complementarity determining region 2(LC CDR2), and/or light chain complementarity determining region 3(LC CDR3) of the CD33 binding domain described herein, and one or more (e.g., all three) heavy chain complementarity determining region 1(HC CDR1), heavy chain complementarity determining region 2(HC CDR2), and heavy chain complementarity determining region 3(HC CDR3) of the CD33 binding domain described herein, e.g., a human or humanized CD33 binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs.

In one embodiment, the encoded CD33 binding domain comprises a light chain variable region as described herein (e.g., SEQ ID NOS: 66, 67, 68, 69, 70, 71, 72, 73, or 74) and/or a heavy chain variable region as described herein (e.g., SEQ ID NOS: 57, 58, 59, 60, 61, 62, 63, 64, or 65). In one embodiment, the encoded CD33 binding domain is an scFv of a light chain and a heavy chain comprising the amino acid sequences of

SEQ ID NO

39, 40, 41, 42, 43, 44, 45, 46 or 47. In one embodiment, the CD33 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of the light chain variable region provided in SEQ ID NO:66, 67, 68, 69, 70, 71, 72, 73 or 74, but NO more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO:66, 67, 68, 69, 70, 71, 72, 73 or 74; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of the heavy chain variable region provided in SEQ ID NO:57, 58, 59, 60, 61, 62, 63, 64 or 65 but NO more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions) or a sequence 95-99% identical to the amino acid sequence of SEQ ID NO:57, 58, 59, 60, 61, 62, 63, 64 or 65. In one embodiment, the CD33 binding domain comprises a sequence selected from the group consisting of SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46 and SEQ ID NO 47 or a sequence 95-99% identical thereto. In one embodiment, the encoded CD33 binding domain is an scFv and the light chain variable region comprising an amino acid sequence described herein (e.g., in table 2) is linked via a linker (e.g., a linker described herein) to the heavy chain variable region comprising an amino acid sequence described herein (e.g., in table 2). In one embodiment, the encoded CD33 binding domain comprises a (Gly4-Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 4(SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can be, for example, in any of the following orientations: a light chain variable region-linker-heavy chain variable region or a heavy chain variable region-linker-light chain variable region.

In another aspect, the invention relates to an isolated CD33 binding domain (e.g., a polypeptide, an antibody, or a fragment thereof) molecule encoded by a nucleic acid molecule. In one embodiment, the isolated CD33 binding domain comprises a sequence selected from the group consisting of SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, and SEQ ID NO 56 or a sequence 95-99% identical thereto.

In another aspect, the invention relates to an isolated Chimeric Antigen Receptor (CAR) molecule (e.g., a polypeptide) comprising a CD33 binding domain (e.g., a human or humanized antibody or antibody fragment that specifically binds to CD 33), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain). In one embodiment, the CAR comprises an antibody or antibody fragment comprising a CD33 binding domain described herein (e.g., a human or humanized antibody or antibody fragment that specifically binds to CD33 as described herein), a transmembrane domain described herein, and an intracellular signaling domain described herein (e.g., an intracellular signaling domain comprising a co-stimulatory domain and/or a primary signaling domain described herein).

In one embodiment, the CD33 binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1(LC CDR1), light chain complementarity determining region 2(LC CDR2), and light chain complementarity determining region 3(LC CDR3) of the CD33 binding domain described herein, and one or more (e.g., all three) heavy chain complementarity determining region 1(HC CDR1), heavy chain complementarity determining region 2(HC CDR2), and/or heavy chain complementarity determining region 3(HC CDR3) of the CD33 binding domain described herein, e.g., a CD33 binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs. In one embodiment, the CD33 binding domain comprises a light chain variable region described herein (e.g., in table 2) and/or a heavy chain variable region described herein (e.g., in table 2 or table 9). In one embodiment, the CD33 binding domain is an scFv comprising a light chain and a heavy chain of the amino acid sequences listed in table 2 or table 9. In one embodiment, the CD33 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of the light chain variable region provided in table 2 or 9, but no more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence having 95-99% identity to the amino acid sequence provided in table 2 or 9; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of the heavy chain variable region provided in table 2 or 9, but no more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence having 95-99% identity to the amino acid sequence provided in table 2 or 9.

In other embodiments, the CD33 binding domain comprises the HC CDR1, HC CDR2, and HC CDR3 of any of the CD33 heavy chain binding domain amino acid sequences listed in table 2 or table 9. In embodiments, the CD33 binding domain further comprises LC CDR1, LC CDR2, and LC CDR 3. In embodiments, the CD33 binding domain comprises LC CDR1, LC CDR2, and LC CDR3 of any of the CD33 light chain binding domain amino acid sequences listed in table 2 or table 9.

In some embodiments, the CD33 binding domain comprises one, two, or all of LC CDR1, LC CDR2, and LC CDR3 of any CD33 light chain binding domain amino acid sequence listed in table 2 or table 9 and one, two, or all of HC CDR1, HC CDR2, and HC CDR3 of any CD33 heavy chain binding domain amino acid sequence listed in table 2 or table 9.

In one embodiment, the CD33 binding domain comprises a sequence selected from the group consisting of SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 57-74 or SEQ ID NO 262-268; or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions) relative to any of the foregoing sequences; or a sequence having 95-99% identity thereto. In one embodiment, the CD33 binding domain is an scFv and the light chain variable region comprising an amino acid sequence described herein (e.g., in table 2 or table 9) is linked via a linker (e.g., a linker described herein) to the heavy chain variable region comprising an amino acid sequence described herein (e.g., in table 2 or table 9). In one embodiment, the CD33 binding domain comprises a (Gly4-Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 4(SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can be, for example, in any of the following orientations: a light chain variable region-linker-heavy chain variable region or a heavy chain variable region-linker-light chain variable region.

In one embodiment, the isolated CAR molecule comprises a transmembrane domain of a protein (e.g., a protein described herein, e.g., selected from the group consisting of the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD 154). In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO 6. In one embodiment, the transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of SEQ ID No. 6, but NO more than 20, 10 or 5 modifications (e.g., substitutions, e.g., conservative substitutions) or a sequence that is 95-99% identical to the amino acid sequence of SEQ ID No. 6.

In one embodiment, the CD33 binding domain is connected to the transmembrane domain by a hinge region (e.g., a hinge region as described herein). In one embodiment, the encoded hinge region comprises SEQ ID NO 2 or a sequence 95-99% identical thereto.

In one embodiment, the isolated CAR molecule further comprises a sequence encoding a co-stimulatory domain (e.g., a co-stimulatory domain described herein).

In embodiments, the intracellular signaling domain of the isolated CAR molecule comprises a costimulatory domain. In embodiments, the intracellular signaling domain of the isolated CAR molecule comprises a primary signaling domain. In embodiments, the intracellular signaling domain of the isolated CAR molecule comprises a costimulatory domain and a primary signaling domain.

In one embodiment, the co-stimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signal molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1(CD11a/CD a), 4-1BB (CD137), B a-H a, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHT TR), KIRDS a, SLAMF a, NKp a (KLRF a), NKp a, CD a alpha, CD a beta, IL2 a gamma, ITIL 7 alpha, IT3672, VLITGA 72, VLITGA a, GAITGB 72, GAITGA a, GAITGB 11-a, GAITGA a, GAITGB 11, GAITGA a, GAITGB a, GAIT11, CD a, GAITGA a, GAITGB 72, GAITGA a, CD a, GAITGB 11-a, GAITGB 11, GAITGA a, GAITGB a, CD a, GAITGA a, GAITGB 11-a, GAITCD a, GAITGA a, CD a, GAITGB 11-a, CD a, GAITGB 11, SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds to CD 83.

In one embodiment, the co-stimulatory domain of 4-1BB comprises the amino acid sequence of SEQ ID NO 7. In one embodiment, the co-stimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions, e.g., conservative substitutions), but NO more than 20, 10 or 5 modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of SEQ ID No. 7 or a sequence that is 95-99% identical to the amino acid sequence of SEQ ID No. 7. In another embodiment, the co-stimulatory domain of CD28 comprises the amino acid sequence of SEQ ID NO: 379. In one embodiment, the co-stimulatory domain comprises an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO:379 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 379. In another embodiment, the CD27 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO 8. In one embodiment, the co-stimulatory domain comprises an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO. 8 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 8. In another embodiment, the costimulatory domain of ICOS comprises the amino acid sequence of SEQ ID NO 381. In one embodiment, the co-stimulatory domain comprises an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO. 381 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 381.

In embodiments, the primary signaling domain comprises a functional signaling domain of CD3 ζ. In embodiments, the functional signaling domain of CD3 ζ comprises SEQ ID NO:9 (mutant CD3 ζ) or SEQ ID NO:10 (wild type human CD3 ζ) or a sequence having 95-99% identity thereto.

In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 ζ. In one embodiment, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO. 7 and/or the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10. In one embodiment, the intracellular signaling domain comprises an amino acid sequence having the amino acid sequence of SEQ ID NO. 7 and/or the sequence of SEQ ID NO. 9 or SEQ ID NO. 10 with at least one, two or three modifications (e.g., substitutions, such as conservative substitutions), but NO more than 20, 10 or 5 modifications (e.g., substitutions, such as conservative substitutions) or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 7 and/or the sequence of SEQ ID NO. 9 or SEQ ID NO. 10. In one embodiment, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO 7 and/or the sequence of SEQ ID NO 9 or SEQ ID NO 10, wherein the sequences comprising the intracellular signaling domain are expressed in frame and as a single polypeptide chain.

In one embodiment, the intracellular signaling domain comprises a functional signaling domain of CD27 and/or a functional signaling domain of CD3 ζ. In one embodiment, the CD27 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO. 8 and/or the CD3 ζ amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10. In one embodiment, the intracellular signaling domain comprises an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications, of the amino acid sequence of SEQ ID NO. 8 and/or of SEQ ID NO. 9 or of SEQ ID NO. 10 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 8 and/or to the amino acid sequence of SEQ ID NO. 9 or of SEQ ID NO. 10. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO 8 and the sequence of SEQ ID NO 9 or SEQ ID NO 10, wherein the sequences comprising the intracellular signaling domain are expressed in frame and as a single polypeptide chain.

In one embodiment, the intracellular signaling domain comprises a functional signaling domain of CD28 and/or a functional signaling domain of CD3 ζ. In one embodiment, the encoded intracellular signaling domain of CD28 comprises the amino acid sequence of SEQ ID NO:379 and/or the amino acid sequence of CD3 ζ of SEQ ID NO:9 or SEQ ID NO: 10. In one embodiment, the intracellular signaling domain comprises an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO:379 or SEQ ID NO:10 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO:379 and/or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO: 10. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO:379 and the sequence of SEQ ID NO:9 or SEQ ID NO:10, wherein the sequences comprising the intracellular signaling domain are expressed in frame and as a single polypeptide chain.

In one embodiment, the intracellular signaling domain comprises a functional signaling domain of ICOS and/or a functional signaling domain of CD3 ζ. In one embodiment, the ICOS intracellular signaling domain comprises the amino acid sequence of SEQ ID NO. 381 and/or the CD3 ζ amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10. In one embodiment, the intracellular signaling domain comprises an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications, of the amino acid sequence of SEQ ID NO. 381 and/or of the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 381 and/or to the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10. In one embodiment, the encoded intracellular signaling domain comprises the sequence of SEQ ID NO:381 and the sequence of SEQ ID NO:9 or SEQ ID NO:10, wherein the sequences comprising the intracellular signaling domain are expressed in frame and as a single polypeptide chain.

In one embodiment, the isolated CAR molecule further comprises a leader sequence (e.g., a leader sequence described herein). In one embodiment, the leader sequence comprises the amino acid sequence of SEQ ID NO. 1 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 1.

In another aspect, the invention relates to an isolated CAR molecule comprising a leader sequence, e.g., a leader sequence described herein (e.g., the leader sequence of SEQ ID NO: 1) or a leader sequence having 95-99% identity thereto; a CD33 binding domain as described herein, e.g., a CD33 binding domain comprising LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 as described herein, e.g., a CD33 binding domain as described in table 2, or a CD33 binding domain having a sequence from 95-99% identity thereto; a hinge region, e.g., a hinge region described herein, e.g., a hinge region of SEQ ID NO. 2, or a hinge region having 95-99% identity thereto; a transmembrane domain, e.g., a transmembrane domain as described herein, e.g., a transmembrane domain having the sequence of SEQ ID No. 6 or a sequence 95-99% identical thereto; an intracellular signaling domain, e.g., an intracellular signaling domain described herein (e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain). In one embodiment, the intracellular signaling domain comprises a costimulatory domain, e.g., a costimulatory domain described herein, e.g., a 4-1BB costimulatory domain having the sequence of SEQ ID NO:7 or 95-99% identity thereto, and/or a primary signaling domain (e.g., a primary signaling domain described herein, e.g., a CD3 zeta stimulatory domain having the sequence of SEQ ID NO:9 or SEQ ID NO:10 or 95-99% identity thereto). In one embodiment, the intracellular signaling domain comprises a costimulatory domain, e.g., a costimulatory domain described herein, e.g., a 4-1BB costimulatory domain having the sequence of SEQ ID NO:7, and/or a primary signaling domain (e.g., a primary signaling domain described herein, e.g., a CD3 zeta stimulatory domain having the sequence of SEQ ID NO:9 or SEQ ID NO: 10).

In one embodiment, the isolated CAR molecule comprises (e.g., consists of) the amino acid sequence of SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, or SEQ ID NO 56 or an amino acid sequence having at least 1, 2, 3, 4, 5, 10, 15, 20 or 30 modifications (e.g., substitutions, e.g., conservative substitutions) but NO more than 60, 50 or 40 modifications (e.g., substitutions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, or SEQ ID NO 56 or an amino acid sequence having at least 1, 2, 3, 4, 5, 10, 15, 20 or 30 modifications (e.g., substitutions, e.g., conservative substitutions), or NO more than 60, 50 or 40 modifications (e.g., conservative substitutions), or a combination of, The amino acid sequences of SEQ ID NO 49, 50, 51, 52, 53, 54, 55 or 56 have 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

In one aspect, the invention relates to a CD33 binding domain, the CD33 binding domain comprising one or more (e.g., all three) light chain complementarity determining region 1(LC CDR1), light chain complementarity determining region 2(LC CDR2), and light chain complementarity determining region 3(LC CDR3) of a CD33 binding domain described herein, and/or one or more (e.g., all three) heavy chain complementarity determining region 1(HC CDR1), heavy chain complementarity determining region 2(HC CDR2), and heavy chain complementarity determining region 3(HC CDR3) of a CD33 binding domain described herein, e.g., a CD33 binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs.

In one embodiment, the CD33 binding domain comprises a light chain variable region as described herein (e.g., SEQ ID NOS: 66, 67, 68, 69, 70, 71, 72, 73, or 74) and/or a heavy chain variable region as described herein (e.g., SEQ ID NOS: 57, 58, 59, 60, 61, 62, 63, 64, or 65). In one embodiment, the CD33 binding domain is an scFv of a light chain and a heavy chain comprising the amino acid sequences of

SEQ ID NOs

39, 40, 41, 42, 43, 44, 45, 46 or 47. In one embodiment, the CD33 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of the light chain variable region provided in SEQ ID NO:66, 67, 68, 69, 70, 71, 72, 73 or 74, but NO more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence 95-99% identical to the amino acid sequence in SEQ ID NO:66, 67, 68, 69, 70, 71, 72, 73 or 74; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of the heavy chain variable region provided in SEQ ID NO:57, 58, 59, 60, 61, 62, 63, 64 or 65, but NO more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence 95-99% identical to the amino acid sequence of SEQ ID NO:57, 58, 59, 60, 61, 62, 63, 64 or 65. In one embodiment, the CD33 binding domain comprises a sequence selected from the group consisting of SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46 and SEQ ID NO 47 or a sequence 95-99% identical thereto. In one embodiment, the CD33 binding domain is an scFv and the light chain variable region comprising an amino acid sequence described herein (e.g., in table 2 or table 9) is linked via a linker (e.g., a linker described herein) to the heavy chain variable region comprising an amino acid sequence described herein (e.g., in table 2 or table 9). In one embodiment, the CD33 binding domain comprises a (Gly4-Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 4(SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can be, for example, in any of the following orientations: a light chain variable region-linker-heavy chain variable region or a heavy chain variable region-linker-light chain variable region.

In another aspect, the invention relates to a vector comprising a nucleic acid molecule described herein, e.g., a nucleic acid molecule encoding a CAR described herein. In one embodiment, the vector is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

In one embodiment, the vector is a lentiviral vector. In one embodiment, the vector further comprises a promoter. In one embodiment, the promoter is the EF-1 promoter. In one embodiment, the EF-1 promoter comprises the sequence of SEQ ID NO 11. In another embodiment, the promoter is a PGK promoter, e.g., a truncated PGK promoter as described herein.

In one embodiment, the vector is an in vitro transcription vector, e.g., a vector that transcribes RNA of a nucleic acid molecule described herein. In one embodiment, the nucleic acid sequence in the vector further comprises a poly A tail, e.g., a poly A tail as described herein, e.g., a poly A tail comprising about 150 adenosine bases (SEQ ID NO: 377). In one embodiment, the nucleic acid sequence in the vector further comprises a 3 'UTR, e.g., a 3' UTR as described herein, e.g., a 3 'UTR comprising at least one repeat of a 3' UTR derived from human β -globin. In one embodiment, the nucleic acid sequence in the vector further comprises a promoter, for example, the T2A promoter.

In another aspect, the invention relates to a cell comprising a vector described herein. In one embodiment, the cell is a cell described herein, e.g., an immune effector cell, e.g., a human T cell described herein, or a human NK cell, e.g., a human NK cell described herein. In one embodiment, the human T cell is a CD8+ T cell.

In one embodiment, a CAR-expressing cell described herein can also express another agent, e.g., an agent that enhances the activity of the CAR-expressing cell. For example, in one embodiment, the substance may be a substance that inhibits inhibitory molecules. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR β, e.g., as described herein. In one embodiment, the substance that inhibits an inhibitory molecule comprises a first polypeptide (e.g., an inhibitory molecule) associated with a second polypeptide (e.g., an intracellular signaling domain described herein) that provides a forward signal to the cell. In one embodiment, the agent comprises a first polypeptide, e.g., an inhibitory molecule such as PD1, PD-L1, PD-L2, LAG3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), CTLA4, VISTA, CD160, BTLA, LAIR1, TIM3, 2B4, TGFR β, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and tig, or a fragment of any of these molecules (e.g., at least a portion of the ectodomain of any of these molecules) and a primary polypeptide, e.g., a signaling domain comprising a costimulatory domain (e.g., CD 41, CD28, CD27, and/or a secondary signaling domain (e.g., a signaling domain of CD 3) as described herein, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD 1) and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein).

In another aspect, the invention relates to a method of making a cell, the method comprising transducing a cell described herein, e.g., an immune effector cell described herein, e.g., a T cell or NK cell described herein, with a vector comprising a nucleic acid encoding a CAR (e.g., a CAR described herein).

The invention also provides a method of producing a population of RNA-engineered cells (e.g., cells described herein, e.g., immune effector cells, e.g., T cells or NK cells) that transiently express an exogenous RNA. The method comprises introducing an in vitro transcribed RNA or a synthetic RNA into the cell, wherein the RNA comprises a nucleic acid encoding a CAR molecule described herein.

In another aspect, the invention relates to a method of providing anti-tumor immunity in a mammal, the method comprising administering to the mammal an effective amount of a cell that expresses a CAR molecule, e.g., a cell that expresses a CAR molecule described herein. In one embodiment, the cell is an autoimmune effector cell, e.g., a T cell or NK cell. In one embodiment, the cell is an allogeneic immune effector cell, e.g., a T cell or NK cell. In one embodiment, the mammal is a human, e.g., a hematological cancer patient.

In another aspect, the invention relates to a method of treating a mammal having a disease associated with expression of CD33 (e.g., a proliferative disease, a precancerous condition, and a non-cancer related indication associated with expression of CD 33), the method comprising administering to the mammal an effective amount of a cell that expresses a CAR molecule (e.g., a CAR molecule described herein). In one embodiment, the mammal is a human, e.g., a hematological cancer patient.

In one embodiment, the disease is a disease described herein. In one embodiment, the disease associated with expression of CD33 is selected from a proliferative disease such as cancer or a malignancy or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or pre-leukemia, or a non-cancer related indication associated with expression of CD 33. In one embodiment, the disease is a hematological cancer selected from one or more acute leukemias, including but not limited to Acute Myeloid Leukemia (AML); myelodysplastic syndrome; myeloproliferative tumors; chronic Myeloid Leukemia (CML); a blast cell plasma cell-like dendritic cell tumor; and include, but are not limited to, diseases associated with expression of CD33, including, but not limited to, atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing CD 33; and combinations thereof. In one embodiment, a cell expressing a CAR molecule (e.g., a CAR molecule described herein) is administered in combination with an agent that increases the efficacy of the cell expressing the CAR molecule (e.g., an agent described herein).

In another aspect, the invention relates to a method of conditioning a subject prior to cell transplantation, comprising administering to the subject an effective amount of a cell comprising a CAR molecule disclosed herein. In one embodiment, the cell transplantation is a stem cell transplantation. The stem cell transplantation is hematopoietic stem cell transplantation or bone marrow transplantation. In one embodiment, the cell transplantation is allogeneic or autologous.

In one embodiment, conditioning the subject prior to cell transplantation comprises reducing the number of cells expressing CD33 in the subject. The cells expressing CD33 in the subject are normal cells expressing CD33 or cancer cells expressing CD33, and in some cases, this conditioning in the subject reduces normal cells expressing CD33 and cancer cells expressing CD33 prior to cell transplantation.

In one embodiment, a cell expressing a CAR molecule (e.g., a CAR molecule described herein) is administered in combination with an immunopotentiating low dose of an mTOR inhibitor. While not wishing to be bound by theory, it is believed that immunopotentiating low doses (e.g., doses insufficient to completely suppress the immune system, but sufficient to improve immune function) treatment is accompanied by a decrease in PD-1 positive immune effector cells (e.g., T cells or NK cells) or an increase in PD-1 negative cells. PD-1 positive immune effector cells (e.g., T cells or NK cells) can be depleted, but not PD-1 negative immune effector cells (e.g., T cells or NK cells), by binding to cells expressing a PD-1 ligand (e.g., PD-L1 or PD-L2).

In one embodiment, such a method can be used to optimize the performance of a CAR cell described herein in a subject. While not wishing to be bound by theory, it is believed that in one embodiment, the expression of endogenous unmodified immune effector cells (e.g., T cells or NK cells) is improved. While not wishing to be bound by theory, it is believed that in one embodiment, the performance of cells expressing CD33 CAR is improved. In other embodiments, cells (e.g., immune effector cells (e.g., T cells or NK cells)) that have been or will be engineered to express a CAR can be treated ex vivo by contacting with an mTOR inhibitor in an amount that increases the number of PD1 negative immune effector cells (e.g., T cells or NK cells) or increases the ratio of PD1 negative immune effector cells (e.g., T cells or NK cells)/PD 1 positive immune effector cells (e.g., T cells or NK cells).

In one embodiment, administration of an immunopotentiating low dose mTOR inhibitor (e.g., an allosteric inhibitor (e.g., RAD001) or a catalytic inhibitor) is initiated prior to administration of a CAR-expressing cell (e.g., an immune effector cell (e.g., a T cell or NK cell)) as described herein. In one embodiment, the CAR cell is administered after a sufficient time for the mTOR inhibitor or sufficient administration of the mTOR inhibitor such that the level of PD 1-negative immune effector cells (e.g., T cells or NK cells) or the ratio of PD 1-negative immune effector cells (e.g., T cells or NK cells)/PD 1-positive immune effector cells (e.g., T cells or NK cells) has been at least transiently increased. In one embodiment, the cells to be engineered to express the CAR, e.g., immune effector cells (e.g., T cells or NK cells), are harvested at a sufficient time or after sufficient administration of an immunopotentiating low dose of an mTOR inhibitor such that the level of PD1 negative immune effector cells (e.g., T cells) or the ratio of PD1 negative immune effector cells (e.g., T cells or NK cells)/PD 1 positive immune effector cells (e.g., T cells or NK cells) in or harvested from the subject has been at least transiently increased.

In one embodiment, the invention provides an mTOR inhibitor for use in treating a subject, wherein the mTOR inhibitor enhances the immune response in the subject, and wherein the subject has received, is receiving, or is about to receive an immune effector cell expressing a CD33 CAR as described herein.

In one embodiment, a cell expressing a CAR molecule (e.g., a CAR molecule described herein) is administered in combination with an agent that ameliorates one or more side effects (e.g., an agent described herein), wherein the side effects are associated with administration of the cell expressing the CAR molecule.

In one embodiment, a cell expressing a CAR molecule (e.g., a CAR molecule described herein) is administered in combination with an agent that treats a CD 33-related disease (e.g., an agent described herein). In certain embodiments, the CD 33-associated disease is a proliferative disease such as cancer or a malignancy or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or pre-leukemia, or is a non-cancer related indication associated with the expression of CD 33.

In certain embodiments, the CD 33-associated disease is a hematological cancer selected from one or more acute leukemias, including but not limited to Acute Myeloid Leukemia (AML); myelodysplastic syndrome; myeloproliferative tumors; chronic Myeloid Leukemia (CML); a blast cell plasma cell-like dendritic cell tumor; and include, but are not limited to, diseases associated with expression of CD33, including, but not limited to, atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing CD 33; and combinations thereof.

In some embodiments, the CD33 CARs described herein target cells expressing CD 33. In embodiments, the CD33 CARs described herein target MDS blast cells. In some embodiments, MDS blasts comprise a 5q deletion (del (5 q)). In embodiments, the cells expressing a CD33CAR described herein are used to treat a subject with MDS. In embodiments, the cells expressing a CD33CAR described herein are used to treat a subject having MDS associated with isolated del (5 q).

In embodiments, the CD33 CARs described herein target MDSCs, e.g., MDSCs in a subject having cancer (e.g., multiple myeloma, chronic lymphocytic leukemia, or a solid malignancy such as ovarian, colon, or breast cancer). In embodiments, the MDSCs are lineage negative (LIN-), HLA-DR negative, and CD33 positive. In some embodiments, the CD33 CAR-expressing cells described herein target MDS blast cells and MDSCs. In embodiments, the CD33 CAR-expressing cells described herein are used to treat multiple myeloma, Chronic Lymphocytic Leukemia (CLL), or a solid malignancy such as ovarian, colon, or breast cancer.

In another aspect, the invention relates to an isolated nucleic acid molecule encoding a CAR of the invention, an isolated polypeptide molecule of a CAR of the invention, a vector comprising a CAR of the invention, and a cell comprising a CAR of the invention, for use as a medicament, e.g., as described herein.

In another aspect, the invention relates to an isolated nucleic acid molecule encoding a CAR of the invention, an isolated polypeptide molecule of a CAR of the invention, a vector comprising a CAR of the invention, and a cell comprising a CAR of the invention, e.g., as described herein, for use in treating a disease that expresses CD33 (e.g., a disease that expresses CD33 as described herein).

Additional features and embodiments of the foregoing compositions and methods include one or more of the following:

in certain embodiments, a CD33 CAR molecule (e.g., a CD33 CAR nucleic acid or a CD33 CAR polypeptide as described herein), or a CD33 binding domain as described herein, comprises 1, 2, or 3 CDRs from a heavy chain variable region (e.g., HC CDR1, HC CDR2, and/or HC CDR3) provided in table 3; and/or 1, 2, or 3 CDRs from a light chain variable region of CAR33-1, CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9 (e.g., LC CDR1, LC CDR2, and/or LC CDR3) provided in table 4; or a sequence that is substantially identical (e.g., 95-99% identical, or up to 5, 4, 3, 2, or 1 amino acid change, e.g., a substitution (e.g., a conservative substitution)) to any of the foregoing sequences.

In certain embodiments, a CD33 CAR molecule (e.g., a CD33 CAR nucleic acid or a CD33 CAR polypeptide as described herein), or an anti-CD 33 antigen binding domain as described herein, comprises 1, 2, or 3 CDRs from a heavy chain variable region (e.g., HC CDR1, HC CDR2, and/or HC CDR3) provided in table 10; and/or 1, 2, or 3 CDRs from a light chain variable region of CAR33-1, CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9 (e.g., LC CDR1, LC CDR2, and/or LC CDR3) provided in table 11; or a sequence that is substantially identical (e.g., 95-99% identical, or up to 5, 4, 3, 2, or 1 amino acid change, e.g., a substitution (e.g., a conservative substitution)) to any of the foregoing sequences.

In certain embodiments, the CD33 CAR molecule or anti-CD 33 antigen binding domain comprises 1, 2, or 3 CDRs from a heavy chain variable region provided in table 12 (e.g., HCDR1, HCDR2, and/or HCDR 3); and/or 1, 2, or 3 CDRs from a light chain variable region of CAR33-1, CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9 (e.g., LC CDR1, LC CDR2, and/or LC CDR3) provided in table 13; or a sequence that is substantially identical (e.g., 95-99% identical, or up to 5, 4, 3, 2, or 1 amino acid change, e.g., a substitution (e.g., a conservative substitution)) to any of the foregoing sequences.

In certain embodiments, the CD33 CAR molecule or anti-CD 33 antigen binding domain comprises

(i) LC CDR1, LC CDR2 and LC CDR3 of any of the CD33 light chain binding domain amino acid sequences listed in table 2 or table 9, or LC CDRs in table 4, table 9, table 11 or table 13; and/or

(ii) The HC CDR1, HC CDR2 and HC CDR3 of any of the CD33 heavy chain binding domain amino acid sequences listed in table 2 or table 9, or the HC CDR in table 3, table 9, table 10 or table 12.

In certain embodiments, a CD33 CAR molecule (e.g., a CD33 CAR nucleic acid or CD33 CAR polypeptide as described herein) or an anti-CD 33 antigen binding domain as described herein comprises:

(1) Three Light Chain (LC) CDRs selected from one of:

(i) LC CDR1 of SEQ ID NO 111, LC CDR2 of SEQ ID NO 120 and LC CDR3 of SEQ ID NO 129 of CAR 33-1;

(ii) LC CDR1 of SEQ ID NO:112, LC CDR2 of SEQ ID NO:121 and LC CDR3 of SEQ ID NO:130 of CAR 33-2;

(iii) LC CDR1 of SEQ ID NO 113, LC CDR2 of SEQ ID NO 122 and LC CDR3 of SEQ ID NO 131 of CAR 33-3;

(iv) LC CDR1 of SEQ ID NO:114, LC CDR2 of SEQ ID NO:123 and LC CDR3 of SEQ ID NO:132 of CAR 33-4;

(iv) LC CDR1 of SEQ ID NO 115, LC CDR2 of SEQ ID NO 124 and LC CDR3 of SEQ ID NO 133 of CAR 33-5;

(vi) LC CDR1 of SEQ ID NO:116, LC CDR2 of SEQ ID NO:125, and LC CDR3 of SEQ ID NO:134 of CAR 33-6;

(vii) LC CDR1 of SEQ ID NO 117, LC CDR2 of SEQ ID NO 126 and LC CDR3 of SEQ ID NO 135 of CAR 33-7;

(viii) LC CDR1 of SEQ ID NO 118, LC CDR2 of SEQ ID NO 127 and LC CDR3 of SEQ ID NO 136 of CAR 33-8; or

(ix) LC CDR1 of SEQ ID NO 119, LC CDR2 of SEQ ID NO 128 and LC CDR3 of SEQ ID NO 137 of CAR 33-9; and/or

(2) Three Heavy Chain (HC) CDRs selected from one of:

(i) HC CDR1 of SEQ ID NO:84, HC CDR2 of SEQ ID NO:93 and HCCDR3 of SEQ ID NO:102 of CAR 33-1;

(ii) HC CDR1 of SEQ ID NO 85, HC CDR2 of SEQ ID NO 94 and HCCDR3 of SEQ ID NO 103 of CAR 33-2;

(iii) HC CDR1 of SEQ ID NO 86, HC CDR2 of SEQ ID NO 95, and HCCDR3 of SEQ ID NO 104 of CAR 33-3;

(iv) HC CDR1 of SEQ ID NO 87, HC CDR2 of SEQ ID NO 96 and HCCDR3 of SEQ ID NO 105 of CAR 33-4;

(iv) HC CDR1 of SEQ ID NO:88, HC CDR2 of SEQ ID NO:97 and HCCDR3 of SEQ ID NO:106 of CAR 33-5;

(vi) HC CDR1 of SEQ ID NO. 89, HC CDR2 of SEQ ID NO. 98 and HCCDR3 of SEQ ID NO. 107 of CAR 33-6;

(vii) HC CDR1 of SEQ ID NO:90, HC CDR2 of SEQ ID NO:99, and HCCDR3 of SEQ ID NO:108 of CAR 33-7;

(viii) HC CDR1 of SEQ ID NO 91, HC CDR2 of SEQ ID NO 100 and HCCDR3 of SEQ ID NO 109 of CAR 33-8; or

(ix) HC CDR1 of SEQ ID NO 92, HC CDR2 of SEQ ID NO 101, and HCCDR3 of SEQ ID NO 110 of CAR 33-9.

(1) three Light Chain (LC) CDRs selected from one of:

(i) LC CDR1 of SEQ ID NO:296, LC CDR2 of SEQ ID NO:305, and LC CDR3 of SEQ ID NO:314 of CAR 33-1;

(ii) LC CDR1 of SEQ ID NO:297, LC CDR2 of SEQ ID NO:306 and LC CDR3 of SEQ ID NO:315 of CAR 33-2;

(iii) LC CDR1 of SEQ ID NO:298, LC CDR2 of SEQ ID NO:307, and LC CDR3 of SEQ ID NO:316 of CAR 33-3;

(iv) LC CDR1 of SEQ ID NO:299, LC CDR2 of SEQ ID NO:308, and LC CDR3 of SEQ ID NO:317 of CAR 33-4;

(iv) LC CDR1 of SEQ ID NO. 300, LC CDR2 of SEQ ID NO. 309, and LC CDR3 of SEQ ID NO. 318 of CAR 33-5;

(vi) LC CDR1 of SEQ ID NO 301, LC CDR2 of SEQ ID NO 310 and LC CDR3 of SEQ ID NO 319 of CAR 33-6;

(vii) LC CDR1 of SEQ ID NO 302, LC CDR2 of SEQ ID NO 311 and LC CDR3 of SEQ ID NO 320 of CAR 33-7;

(viii) LC CDR1 of SEQ ID NO 303, LC CDR2 of SEQ ID NO 312 and LC CDR3 of SEQ ID NO 321 of CAR 33-8; or

(ix) LC CDR1 of SEQ ID NO:304, LC CDR2 of SEQ ID NO:313, and LC CDR3 of SEQ ID NO:322 of CAR 33-9; and/or

(2) Three Heavy Chain (HC) CDRs selected from one of:

(i) HC CDR1 of SEQ ID NO:269, HC CDR2 of SEQ ID NO:278, and HCCDR3 of SEQ ID NO:287 of CAR 33-1;

(ii) HC CDR1 of SEQ ID NO:270, HC CDR2 of SEQ ID NO:279, and HCCDR3 of SEQ ID NO:288 of CAR 33-2;

(iii) HC CDR1 of SEQ ID NO:271, HC CDR2 of SEQ ID NO:280 and HCCDR3 of SEQ ID NO:289 of CAR 33-3;

(iv) HC CDR1 of SEQ ID NO:272, HC CDR2 of SEQ ID NO:281, and HCCDR3 of SEQ ID NO:290 of CAR 33-4;

(iv) HC CDR1 of SEQ ID NO:273, HC CDR2 of SEQ ID NO:282, and HCCDR3 of SEQ ID NO:291 of CAR 33-5;

(vi) HC CDR1 of SEQ ID NO:274, HC CDR2 of SEQ ID NO:283, and HCCDR3 of SEQ ID NO:292 of CAR 33-6;

(vii) HC CDR1 of SEQ ID NO:275, HC CDR2 of SEQ ID NO:284, and HCCDR3 of SEQ ID NO:293 of CAR 33-7;

(viii) HC CDR1 of SEQ ID NO:276, HC CDR2 of SEQ ID NO:285 and HCCDR3 of SEQ ID NO:294 of CAR 33-8; or

(ix) HC CDR1 of SEQ ID NO 277, HC CDR2 of SEQ ID NO 286, and HCCDR3 of SEQ ID NO 295 of CAR 33-9.

(1) three Light Chain (LC) CDRs selected from one of:

(i) LC CDR1 of SEQ ID NO:350, LC CDR2 of SEQ ID NO:359, and LC CDR3 of SEQ ID NO:368 of CAR 33-1;

(ii) LC CDR1 of SEQ ID NO:351, LC CDR2 of SEQ ID NO:360 and LC CDR3 of SEQ ID NO:369 of CAR 33-2;

(iii) LC CDR1 of SEQ ID NO:352, LC CDR2 of SEQ ID NO:361 and LC CDR3 of SEQ ID NO:370 of CAR 33-3;

(iv) LC CDR1 of SEQ ID NO:353, LC CDR2 of SEQ ID NO:362 and LC CDR3 of SEQ ID NO:371 of CAR 33-4;

(iv) LC CDR1 of SEQ ID NO:354, LC CDR2 of SEQ ID NO:363 of CAR33-5 and LC CDR3 of SEQ ID NO: 372;

(vi) LC CDR1 of SEQ ID NO 355, LC CDR2 of SEQ ID NO 364 and LC CDR3 of SEQ ID NO 373 of CAR 33-6;

(vii) LC CDR1 of SEQ ID NO:356, LC CDR2 of SEQ ID NO:365 and LC CDR3 of SEQ ID NO:374 of CAR 33-7;

(viii) LC CDR1 of SEQ ID NO. 357, LC CDR2 of SEQ ID NO. 366, and LC CDR3 of SEQ ID NO. 375 of CAR 33-8; or

(ix) LC CDR1 of SEQ ID NO:358, LC CDR2 of SEQ ID NO:367, and LC CDR3 of SEQ ID NO:376 of CAR 33-9; and/or

(2) Three Heavy Chain (HC) CDRs selected from one of:

(i) HC CDR1 of SEQ ID NO:323, HC CDR2 of SEQ ID NO:332 and HC CDR3 of SEQ ID NO:341 of CAR 33-1;

(ii) HC CDR1 of SEQ ID NO:324, HC CDR2 of SEQ ID NO:333, and HC CDR3 of SEQ ID NO:342 of CAR 33-2;

(iii) HC CDR1 of SEQ ID NO:325, HC CDR2 of SEQ ID NO:334 and HC CDR3 of SEQ ID NO:343 of CAR 33-3;

(iv) HC CDR1 of SEQ ID NO:326, HC CDR2 of SEQ ID NO:335, and HC CDR3 of SEQ ID NO:344 of CAR 33-4;

(iv) HC CDR1 of SEQ ID NO:327, HC CDR2 of SEQ ID NO:336 and HC CDR3 of SEQ ID NO:345 of CAR 33-5;

(vi) HC CDR1 of SEQ ID NO:328, HC CDR2 of SEQ ID NO:337, and HC CDR3 of SEQ ID NO:346 of CAR 33-6;

(vii) HC CDR1 of SEQ ID NO:329, HC CDR2 of SEQ ID NO:338, and HC CDR3 of SEQ ID NO:347 of CAR 33-7;

(viii) HC CDR1 of SEQ ID NO:330, HC CDR2 of SEQ ID NO:339 and HC CDR3 of SEQ ID NO:348 of CAR 33-8; or

(ix) HC CDR1 of SEQ ID NO:331, HC CDR2 of SEQ ID NO:340, and HC CDR3 of SEQ ID NO:349 of CAR 33-9.

In certain embodiments, a CD33 CAR molecule (e.g., a CD33 CAR nucleic acid or CD33 CAR polypeptide as described herein) or an anti-CD 33 antigen binding domain as described herein comprises: 2213scFv amino acid sequence (SEQ ID NO:142) or a nucleotide sequence encoding 2213scFv (SEQ ID NO:141) or an antigen-binding domain thereof (e.g., the VH, VL or one or more CDRs thereof).

In certain embodiments, a CD33 CAR molecule (e.g., a CD33 CAR nucleic acid or CD33 CAR polypeptide as described herein) or an anti-CD 33 antigen binding domain as described herein comprises: my96 scFv amino acid sequence (SEQ ID NO:147) or an antigen binding domain thereof (e.g., a VH, VL or one or more CDRs thereof).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are now described. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Title, subtitle, or number or letter number elements, such as (a), b), (i), etc., are shown for ease of reading only. The use of title or number or alpha-numbered elements in this document does not require that the steps or elements be performed in alphabetical order and that the steps or elements be necessarily independent of each other. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Brief Description of Drawings

The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1 is an image showing CD33 expression on a majority of blast cells in many primary AML patient samples (using standard side scatter)^{Is low in}CD45^DarknessCharacterized by gating AML blasts); n-each of 35-46.

Fig. 2A, 2B, and 2C are graphs and flow cytometric maps showing the expression of CD33 in bone marrow from myelodysplastic syndrome patients. FIG. 2A shows the percentage of CD 33-expressing cells in the CD34+ CD 38-hematopoietic stem cell compartment of MDS patients. Figure 2B shows the percentage of cells expressing CD33 in the CD34+ CD38+ compartment containing myeloid progenitor cells in MDS patients. FIG. 2C is a histogram showing mean fluorescence intensity from MDS patients in the CD34+ CD 38-compartment.

Figure 3 shows a schematic diagram of the CAR construct used in example 1. All are second generation CARs using 41BB and CD3 zeta signaling. The scFv of CART33 was derived from clone MY 9-6.

Figure 4 is a graph showing the in vitro activity of CART 33. CART 33-mediated T cell degranulation: CAR33 transduced and non-transduced T cells were incubated with CD33+ cell line MOLM14 and control ALL cell line NALM6 in the presence of CD28, CD49d and monensin for 4 hours. CD107a degranulation was measured by flow cytometry. Expression of both the murine CART33 construct and the humanized CART33 construct elicited specific degranulation in the presence of MOLM14 (P < 0.001).

Figure 5 is a graph showing the in vitro activity of CART 33. Cytokine production: both humanized and murine CAR33 expressing T cells produced cytokines upon incubation with MOLM 14. T cells were incubated with CD33+ cell line MOLM14 and control cell line NALM6 for 4 hours. The cells are then fixed, permeabilized and stained for intracellular tumor necrosis factor alpha and interferon gamma. The samples were then analyzed by flow cytometry.

Figure 6 is a graph showing the in vitro activity of CART 33. Proliferation of CAR123 expressing T cells and CAR33 expressing T cells: humanized CART33 and murine CART33 proliferated in response to MOLM 14. T cells were labeled with CFSE and incubated for 120 hours under control conditions or with MOLM14, and CFSE dilutions were measured by flow cytometry as markers of proliferation.

Fig. 7A and 7B are two graphs showing the in vitro activity of CART 33. Specific killing of CAR123-T cells, humanized CAR33-T cells, and murine CAR33 expressing T cells: t cells were incubated with MOLM14 or T cell ALL cell line Jurkat (control) for 24 hours. At low E: T ratios, huCART33 caused significantly more specific killing than murine CART 33. In FIG. 7B, CART33-Jurkat is represented by triangles, CART33-MOLM14 is represented by inverted triangles and CART123-MOLM14 is represented by squares.

FIG. 8 is a graph showing that CART33(IgG4 hinge) and CART33(CD8 hinge) have equivalent in vitro activity. Degranulation assay: CART33(IgG4 hinge), CART33(CD8 hinge), CART123 and non-transduced T cells were incubated with the CD33+ cell line MOLM14 and CD107a degranulation was measured by flow cytometry. Both CART33 constructs underwent specific degranulation in the presence of MOLM 14.

FIG. 9 is a graph showing that CART33(IgG4 hinge) and CART33(CD8 hinge) have equivalent in vitro activity. Cytokine production: after incubation with MOLM14 cell line, both CAR33 construct and CAR123 specifically induced cytokine production. T cells were incubated with MOLM14 in the presence of CD28, CD49d and monensin for 4 hours. Cells were subsequently harvested, fixed, permeabilized and stained for tumor necrosis alpha, MIP1a and interferon gamma. The percentage of cytokine-producing cells was then measured by flow cytometry.

FIG. 10 is a graph showing that CART33(IgG4 hinge) and CART33(CD8 hinge) have equivalent in vitro activity. Untransduced controls, CAR33(IgG4 hinge) (i.e., CAR33-IgG4H) -expressing T cells, CAR33(CD8 hinge) (i.e., CAR33-CD8H) -expressing T cells, or CAR 123-expressing T cells proliferated in response to MOLM 14. T cells were labeled with CFSE and incubated with MOLM14 for 120 hours.

FIG. 11 is a schematic diagram showing a comparison of the anti-tumor effects of CART33-CD8H, CART33-IgG4H and CART123 in vivo. NOD-SCID-common gamma chain knock-out (NSG) mice were injected intravenously with 1x10⁶Individual AML cell lines MOLM14 and the implantation was imaged 6 days later. On day 7, mice were treated with T cells expressing CAR33(IgG4 hinge), CAR33(CD8 hinge), CAR123, or control vectors (non-transduced cells). The total number of injected T cells was 2x10⁶And i.v. Every weekMice were followed by continuous imaging to assess tumor burden.

FIG. 12 is an image showing the equivalent in vivo anti-tumor effects of CART33-CD8H, CART33-IgG4H, and CART123T cells. Displaying tumor burden over time according to bioluminescence imaging (BLI); data were from one experiment (5 per group) and a representative of 4 independent mouse experiments is shown in figure 11.

Figure 13 is a schematic showing a comparison of CART33 and CART123 for the eradication of primary AML in vivo. Primary AML samples were tested at 5x10 ⁶Individual cells were injected intravenously into human cytokine IL3/GM-CSF/SCF transgenic NSG mice (NSGS mice). Implantation was confirmed by retro-orbital bleeding after 2-4 weeks and mice were subsequently treated with CART33, CART123, or control vehicle (untransduced cells). The total number of injected T cells was 1x10⁵And i.v. Mice were followed by a series of retro-orbital bleeds to assess AML burden.

Figure 14 is a graph showing CART33 and CART123 produce equivalent primary AML eradication in vivo. Peripheral blood from Untransduced (UTD), CART33 or CART123 treated mice was analyzed at baseline, day 14 and day + 70. AML according to the experimental set-up described in figure 13 was not detected in mice treated with CART33 or CART 123.

Figure 15 is a graph showing CART33 and CART123 produce equivalent primary AML eradication in vivo. Summary of disease burden measured as blast cells/ul from retro-orbital bleeds from different time points shown from mice in the experimental setup described in fig. 13.

Figure 16 is a graph showing CART33 and CART123 produce equivalent primary AML eradication in vivo. According to the experimental setup described in fig. 13, survival of mice treated with CART33, CART123, or UTD (p <0.001 when treated with CART33 or CART123 compared to UTD).

Fig. 17 is a schematic diagram showing the setup to test the hematopoietic stem cell toxicity of CART33 cells. The experimental scheme is as follows: blood was collected retro-orbitally from Humanized Immune System (HIS) mice 6-8 weeks after injection of human CD34+ cells derived from fetal liver to confirm engraftment of human cells. The mice were then administered CART33 or UTD (1 x10 each)⁶Individual cells) and weekly continuous retro-orbital blood sampling. Then at the secondMice were euthanized on day 28 and organs were harvested and analyzed.

Fig. 18 is a graph showing hematopoietic stem cell toxicity of CART33 cells. Peripheral blood was analyzed by flow cytometry (by retroorbital blood sampling) from day 28 upon completion of the experiment shown in fig. 17. CART33 treatment resulted in a significant reduction in peripheral myeloid cells and monocytes compared to non-transduced T cell treatment.

Fig. 19 is a graph showing hematopoietic stem cell toxicity of CART33 cells. Summary statistics of peripheral blood analysis on day 28 from CART33, UTD treated or untreated mice (n-5) in the experiments shown in figure 17. CART33 on monocytes and CD33⁺Myeloid lineage cells produce significant toxicity, while B cells and platelets are relatively rare.

Fig. 20 is a graph showing hematopoietic stem cell cytotoxicity of CART33 cells. Bone marrow plots were analyzed by flow cytometry at day 28 of mice from the experiment shown in figure 17. CART33 treatment resulted in a significant reduction in individual, huCD45 dark, lineage negative gated myeloid progenitor cells (CD34+ CD38+) and hematopoietic stem cells (CD34+ CD 38-).

Fig. 21 is a graph showing hematopoietic stem cell toxicity of CART33 cells. Sections of femurs from mice from the experiments shown in figure 17 were taken from mice on day 28 after UTD T cell or CART33 cell treatment. huCD45 and CD34 staining was performed by IHC. There was no difference in huCD45 between control T cells and CART33, although both groups showed less huCD45 staining, perhaps consistent with an allogeneic human-anti-human effect. There was a specific reduction of CD34+ cells in CART33 treated mice. The results are representative of two experiments.

Fig. 22 is a schematic diagram showing the setup to test the hematopoietic toxicity of CART33 and CART123 in vivo. An experimental scheme; NSGS mice received intraperitoneal administration of busulfan followed by the next day 2x10 from normal donors⁶Individual T cell depleted bone marrow cells. Implantation was confirmed by flow cytometric analysis of peripheral blood after 4 weeks, and subsequently with 1x10⁶Mice were treated with autologous T cells treated with CART33, CART123, or UTD. Mice were then followed by retroorbital bleeds on

days

7 and 14 and euthanized for necropsy on day 14And (4) performing body dissection.

Figure 23 is a graph showing that CART33 and CART123 produce equivalent hematopoietic toxicity in vivo. A representative plot of bone marrow analyzed by flow cytometry at day 28 of mice from the experiment shown in figure 22 is shown. CART33 and CART123 treatments resulted in a significant reduction in huCD45 dark, Lin-gated myeloid progenitor cells (CD34+ CD38+) and hematopoietic stem cells (CD34+ CD 38-). The results are representative of two experiments.

FIG. 24 is an image showing CART33 and CART123 are cytotoxic myelodysplastic syndrome (MDS) bone marrow cells. CD 34-enriched BM from MDS patients were incubated with UTD, CART33(IgG4 hinge), CART33(CD8 hinge), or CART 123. CD45 dark CD34+ cells were reduced in CART33 or CART123 treated samples.

FIG. 25 is a schematic of a vector expressing murine CART 33.

Figure 26 is a schematic of a vector expressing humanized CART 33.

Figure 27 is an image depicting cell surface expression of scFv on Jurkat T cell line containing a luciferase reporter driven by NFAT regulated promoter (referred to as JNL cells). JNL cells were transduced with lentiviral vectors expressing cDNA encoding GFP, scFv binding to CD19, or cDNA encoding scFv generated against hsCD 33. Cell surface expression of individual scfvs on JNL was detected by incubating cells with Fc-tagged recombinant hsCD33, followed by an Fc-specific secondary antibody conjugated to phycoerythrin.

Fig. 28A, 28B, 28C, and 28D are images showing the ability of individual scfvs targeting hsCD33 to stimulate NFAT activity in JNL cells. JNL cells expressing scFv to hsCD33 were co-cultured with MOLM13 or MOLP8 cell lines expressing hsCD33 or lacking hsCD33 cell surface expression, respectively (FIG. 28A; hsCD33, green solid line; isotype control, grey dashed and shaded area). Figure 28B depicts activation of JNL cells expressing scFv targeting hsCD33 in the presence of MOLM13 cells (solid line) or MOLP8 cells (dashed line). JNL cells expressing the respective scFv were plated at different effector (i.e., JNL cells) to target (i.e., MOLM13 or MOLP8) ratios and Bright-Glo was used on an EnVision instrument 24 hours after incubation ^TMLuciferase assays analyze expression relative to luciferase units (RLU). Figure 28C is a version of figure 28B depicting activation of JNL cells in the presence of MOLM 13. Figure 28D is a version of figure 28B depicting activation of JNL cells in the presence of MOLP 8.

Fig. 29A and 29B are a set of images depicting the activity of scFv targeting hsCD33 in donor-derived primary T cells. The expression of scFv on the cell surface of human primary T cells transduced with lentiviral vectors expressing scFv targeting hscD33 is described. Expression of scFv was detected by incubating the cells with recombinant Fc-tagged hsCD33 and an Fc-specific secondary antibody conjugated to phycoerythrin as described in figure 27.

Fig. 30A and 30B are images depicting proliferative activity of T cells expressing scFv targeting CD 33. T cells were labeled with CFSE and co-cultured or cultured alone (fig. 30A, shaded bars) in the presence of MOLM13 (fig. 30A, solid black line), MOLP8 (fig. 30A, open white bars) to assess the proliferative capacity of UTD primary T cells or cells expressing scFv targeting hsCD 33. Furthermore, antigen-driven cell division in primary T cells was assessed by measuring the Median Fluorescence Intensity (MFI) of CFSE-labeled T cells co-cultured with MOLM13 cells, MOLP8 cells, or alone, which expressed scFv clones CD33-2, -3, -4, -5, -6, and-9 (fig. 30B).

Figure 31 is a graph depicting cytolytic activity of T cells expressing scFV targeted to CD 33. To assess cytolytic activity, 25,000 MOLM13 cells were plated at different effector (i.e., T cell) to target (i.e., MOLM13) ratios along with primary T cells expressing the respective scfvs and the extent of killing by MOLM13 was analyzed by counting the absolute number of CFSE-labeled MOLM13 cells after 4 days of culture.

Figure 32 is an image depicting cross-reactivity of T cells expressing scFv against cynomolgus monkey CD33(cyCD33) targeted to CD33, according to flow cytometry. JNL cells transduced with lentiviral vectors expressing scFv to hsCD33 were incubated with recombinant Fc-tagged hsCD33 (dotted line) or cyCD33 (solid line) followed by an Fc-specific secondary antibody conjugated to phycoerythrin.

Fig. 33A and 33B depict expression of mRNA CAR33 in T cells from normal donors after electroporation. Figure 33A is a series of flow cytometry plots showing CAR33 expression in T cell populations at the time points shown. The percentage of cells expressing CAR33 (boxed) was quantified and shown in the map. Figure 33B is a graphical representation of the percentage expression of CAR 33.

Figures 34A and 34B compare the expression of lentivirus transduced CAR33 with mRNA electroporated CAR 33. Figure 34A shows stable expression of lentivirus transduced CAR33(CART33LV) over the 4 day range at the times indicated. Figure 34B shows transient expression of mRNA electroporated CAR33(CART33RNA) over the 4 day range at the times indicated. Expression of CAR33 is represented by Mean Fluorescence Intensity (MFI) on the x-axis, and total cell number on the y-axis.

Fig. 35A, 35B, 35C, and 35D are graphs comparing cytolytic activity of T cells expressing CD33 following lentiviral transduction or mRNA electroporation. The experiments were repeated 1 day (fig. 35A), 2 days (fig. 35B), 3 days (fig. 35C) and 4 days (fig. 35D) after T cell electroporation. CART33 cells were incubated with the CD33 positive cell line MOLM14 and the control mantle cell lymphoma cell line JEKO at the E: T (effector to target) ratio shown on the x-axis. The percent kill at each ratio is shown on the y-axis.

Fig. 36A and 36B are graphs comparing cytolytic activity over time of T cells expressing CD33 following lentiviral transduction or mRNA electroporation. Figure 36A shows the specific killing effect of lentiviral transduced CAR33 cells compared to RNA CAR33 cells at an E: T (effector: target) ratio of 2:1 incubated for 4 days with MOLM14 cells. Figure 36B shows specific killing of lentiviral transduced CAR33 cells compared to RNA CAR33 cells at an E: T (effector: target) ratio of 1:1 incubated for 4 days with MOLM14 cells.

Fig. 37A, 37B, 37C, and 37D show that CART33 cells displayed robust in vitro effector function in response to the CD33+ cell line MOLM14 or to primary AML samples. The graph represents four independent experiments. Fig. 37A shows CD107A degranulation. CART33, CART123 and non-transduced T cells (UTD) were incubated with the CD33+/CD123+ cell line MOLM14, PMA/ionomycin as a positive non-specific T cell stimulator and the control T cell ALL cell line Jurkat in the presence of CD49d, CD28 co-stimulatory molecule and monensin. CD107a degranulation was measured by flow cytometry after 4 hours incubation. Figure 37B shows specific killing of cells expressing CD 33. CART33, CART123 and UTD were incubated with MOLM14-luc or Jurkat-luc for 24 hours at different E: T ratios as indicated and subsequently bioluminescent imaging was performed as a measure of residual viable cells. Black/solid line (squares) represents CART123 incubated with MOLM 14; blue/dotted line (triangle pointing down/filled triangle) represents CART33 incubated with MOLM 14; and red/dashed lines (triangles pointing up/open triangles) represent CART33 incubated with Jurkat. Fig. 37C and 37D show that CART33 cells proliferated in response to cells expressing CD 33. T cells were labeled with CFSE and incubated for 120 hours with MOLM14, PMA/IONO as positive non-specific T cell stimulator, Jurkat or AML samples as negative controls. The number of T cells proliferating in response to MOLM14 was significantly higher compared to Jurkat and comparable to CART 33.

Fig. 38A, 38B, and 38C show that CART33 cells produce cytokines in response to MOLM14, a cell expressing CD 33. CART33, CART123 and UTD cells were incubated with MOLM14, PMA/ionomycin and Jukat for 4 hours. Cells were then fixed and permeabilized, stained for 5 different cytokines (tumor necrosis factor α, interferon γ, granulocyte macrophage colony stimulating factor, macrophage inflammatory protein 1b, and interleukin-2), and flow cytometric analysis was performed. Most CART33 cells produced more than one cytokine in response to MOLM14 (fig. 38A), similar to their response to PMA/ionomycin (positive control, fig. 38B). FIG. 38C shows that IL-2, IFN- γ, GM-CSF, and TNF- α production in CART33 in response to MOLM14 was significantly higher than CART 123. CART33, CART123 and UTD cells were incubated with MOLM14, Jurkat and PMA/ionomycin for 24 hours. Supernatants were then harvested and subjected to the 30-plex Luminex assay. The levels of the remaining cytokines are shown in figure 39.

Figure 39 is a series of graphs showing comparison of CART33 cells and CART123 cells producing cytokines in response to MOLM 14. CART33, CART123 and UTD cells were incubated with MOLM14, Jurkat and PMA/ionomycin for 24 hours. Supernatants were then harvested and the indicated cytokines were subjected to the 30-plex Luminex assay.

FIG. 40 shows the in vitro specific killing effect of MOLM14 cells expressing CD 33. CART123, CART33(CD8 hinge) and CART33(IgG4 hinge) were incubated with MOLM14 at the indicated E: T ratios and killing was assessed by bioluminescence imaging. At lower E: T ratios, CART33(IgG4 hinge) produced more specific killing than CART33(CD8 hinge).

FIG. 41A, FIG. 41B, and FIG. 41C show anti-tumor activity in myelodysplastic syndrome (MDS). Figure 41A is a graph showing specific CD107a degranulation in response to bone marrow cells from MDS patients. Figure 41B is a set of images showing specific killing of MDS clones with 5q deletions. Fig. 41C is a graph showing quantification of 5q deletion clones remaining after treatment as determined by FISH. There was a significant reduction in the percentage of 5 q-clones in the CART33 treated group when compared to the UTD group and the untreated group.

Fig. 42A, 42B, 42C show CART33 treatment and survival results from MOLM14 implanted xenografts. An experimental protocol is shown in fig. 11. In fig. 42A, tumor burden was quantified over time according to bioluminescence imaging (BLI), data from one experiment (n-5 per group), with each mouse represented by a line. Figure 42B shows the composite survival of three independent experiments. Treatment with CART33 produced a significant survival advantage when compared to UTD treatment. Fig. 42C is a representative bioluminescence image from one experiment.

Fig. 43A and 43B show that CART33 treatment resulted in a dose-dependent reduction in leukemia burden in MOLM14 implanted xenografts. Fig. 43A is a schematic diagram showing the experimental setup described in example 6. Fig. 43B shows quantification of tumor burden as measured by bioluminescence imaging (BLI) over time in different groups.

Fig. 44 is a graph showing tumor burden as measured by bioluminescence imaging (BLI) over time in different groups under the experimental setup shown in fig. 11.

FIG. 45 shows that the combination of RNA-CART33 and chemotherapy results in a further reduction of leukemic burden in MOLM14 implanted xenografts.

Fig. 46A and 46B show antibody binding ability to CD33 and CD123 on MOLM14 and primary AML samples for in vivo experiments. The assay was performed using the QUANTUM SIMPLY CELLULAR kit (Bangs Laboratories, Inc). The samples were washed in flow buffer and then stained with indicated antibodies (CD33 or CD123) conjugated to PE. Five different microspheres provided in the kit were also stained with the same antibody. The mean fluorescence intensity of the target was compared to five microspheres and antibody binding capacity values were then calculated according to the manufacturer's protocol. Fig. 46A shows the antibody binding capacity to CD33 and CD123 of MOLM14, while fig. 46B shows the antibody binding capacity to CD33 and CD123 of the primary samples used in these experiments.

FIG. 47 is a graph showing proliferation in terms of cell counts of various T cells-CART-33T cells (CD33-1 to CD33-9, or Upenn), CART-CD19T cells (CD19), or untransduced T cells (cells) when exposed to PL21, HL-60, or MOLP8 target cells.

FIG. 48A is a graph showing the percentage of lysis of HL-60-Luc target cells when exposed to various CART-33T cells (CD33-1 to CD33-9, or Upenn), CART-CD19T cells (CD19), or untransduced T cells (cells) at various effector to T cell ratios. FIG. 48B is a graph showing the percentage of PL21/Luc target cells lysed upon exposure to various CART-33T cells (CD33-1 to CD33-9, or Upenn), CART-CD19T cells (CD19), or non-transduced T cells (cells) at various effector to T cell ratios. FIG. 48C is a graph showing the percentage of U87/Luc target cells lysed upon exposure to various CART-33T cells (CD33-1 to CD33-9, or Upenn), CART-CD19T cells (CD19), or non-transduced T cells (cells) at various effector to T cell ratios.

FIG. 49 is a graph showing the concentration of cytokines (human interferon- γ (IFN- γ), human interleukin-2 (IL-2), and human Tumor Necrosis Factor (TNF)) produced by various CART-33T cells (CD33-1 to CD33-9, or Upenn), CART-CD19T cells (CD19), or untransduced T cells (cells) when exposed to PL21, HL60, or MOLP8 target cells.

Fig. 50A is a flow cytometry plot showing gating strategy for MDSCs. FIG. 50B is a graph showing the percentage of lineage negative, HLA-DR negative, CD33+ (LIN-HLDR-CD33+) cells in bone marrow from a normal donor (ND BM) or from a myelodysplastic syndrome (MDS BM) patient. Figure 50C is a graph showing the level of CD33 measured in terms of Mean Fluorescence Intensity (MFI) in MDSC populations (MDSCs) compared to malignant MDS populations (MDSs) and normal donor populations (ND-BM).

FIG. 51A is a set of flow cytometry plots showing the degree of degranulation (CD107a levels) and cytokine production (GM-CSF, IL-2, TNF- α) from CART 33. CD107a degranulation and cytokine production are shown on the y-axis, and anti-CD 33CAR is shown on the x-axis. Negative controls (Jurkat) are shown on the left and MDSCs are shown on the right. FIG. 51B is a graph showing the degranulation and cytokine production quantification of CART33 for various targets-Jurkat, PMA/ionomycin (PMA/IONO), MDS (non-MDSC), and MDSC.

Fig. 52A-52D show various configurations on a single vector, e.g., where a U6-regulated shRNA is located upstream or downstream of a EF1 α -regulated CAR-encoding element. In the exemplary constructs depicted in fig. 52A and 52B, transcription occurred through the U6 and EF 1a promoters in the same orientation. In the exemplary constructs plotted in fig. 52C and 52D, transcription occurred through the U6 and EF 1a promoters in different orientations. In fig. 52E, the shRNA (and corresponding U6 promoter) was on the first vector and the CAR (and corresponding EF 1a promoter) was on the second vector.

FIG. 53 depicts the structure of two exemplary RCAR layouts. The antigen binding member comprises an antigen binding domain, a transmembrane domain, and a switch domain. The intracellular binding member comprises a switch domain, a costimulatory signal domain, and a primary signal domain. These two arrangements show that the first and second switch domains described herein can be in different orientations relative to the antigen binding member and intracellular binding member. Other RCAR layouts are further described herein.

Figure 54 shows enhancement of proliferation of CAR-expressing transduced T cells by low dose RAD001 in a cell culture system. CART was co-cultured with Nalm-6 cells in the presence of different concentrations of RAD 001. The number of CAR-positive CD 3-positive T cells (black) and total T cells (grey) was assessed after 4 days of co-culture.

FIG. 55 depicts tumor growth measurements of NALM6-luc cells daily at 0.3, 1, 3, and 10mg/kg (mpk) of RAD001 or vehicle. Circles represent vehicles; squares represent 10mg/kg doses of RAD 001; triangles represent a 3mg/kg dose of RAD001, inverted triangles represent a 1mg/kg dose of RAD 001; and diamonds represent 0.3mg/kg doses of RAD 001.

Fig. 56A and 56B show pharmacokinetic curves showing the amount of RAD001 in the blood of NSG mice bearing NALM6 tumor. Fig. 56A shows day 0 PK after initial dose of RAD 001. Fig. 56B shows the 14 th day PK after end dose RAD 001. Diamonds represent 10mg/kg doses of RAD 001; squares represent 1mg/kg doses of RAD 001; triangles represent 3mg/kg doses of RAD 001; and x' represents a 10mg/kg dose of RAD 001.

Fig. 57A and 57B show in vivo proliferation of humanized CD19 CART cells with or without administration of RAD 001. Low daily doses of RAD001(0.003mg/kg) resulted in enhanced CAR T cell proliferation, above the normal level of huCAR19 proliferation. FIG. 57A shows CD4⁺A CAR T cell; FIG. 57B shows CD8⁺CAR T cells. Circles represent PBS; squares represent huCTL 019; the triangles represent huCTL01 with RAD0019 of 3 mg/kg; the inverted triangle indicates huCTL019 with RAD001 of 0.3 mg/kg; diamonds represent huCTL019 with RAD001 of 0.03 mg/kg; and the circle represents huCTL019 with RAD001 of 0.003 mg/kg.

FIG. 58 is a graph showing progression of HL-60-luc xenograft AML disease. Blue circle: mice treated with 100ul PBS via tail vein; red squares: mice treated with CD19 CAR T cells; green triangle: mice treated with CD33-1 CAR transduced T cells; purple inverted triangle: mice treated with CD33-2CAR transduced T cells; orange diamond shape: mice treated with CD33-4CAR transduced T cells; black squares: mice treated with CD33-5 CAR-transduced T cells; brown triangle: mice treated with CD33-6CAR transduced T cells; dark blue circle: mice treated with CD33-7CAR transduced T cells; and dark purple inverted triangle: mice treated with CD33-9 CAR transduced T cells.

Detailed Description

Definition of

Unless otherwise defined, all terms and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

The term "about" when referring to a measurable value such as a quantity, time duration, or the like, is intended to encompass a variation of ± 20% or in some cases ± 10% or in some cases ± 5% or in some cases ± 1% or in some cases ± 0.1% from the specified value, as such variations are suitable for carrying out the disclosed methods.

The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the domains in the CAR polypeptide construct are in the same polypeptide chain, e.g., comprise a chimeric fusion protein. In some embodiments, the domains in the CAR polypeptide construct are not contiguous with each other, e.g., in different polypeptide chains, e.g., as provided in the RCAR as described herein.

In one aspect, the stimulatory molecule of the CAR is a zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., the primary signaling domain of CD 3-zeta). In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one aspect, the co-stimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, ICOS and/or CD 28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cell processing and localization of the CAR to the cell membrane.

A CAR comprising an antigen binding domain (e.g., scFv, single domain antibody, or TCR (e.g., TCR a binding domain or TCR β binding domain)) that targets a specific tumor marker X, where X can be a tumor marker as described herein, is also referred to as XCAR. For example, a CAR comprising an antigen binding domain that targets CD33 is referred to as a CD33 CAR. The CAR can be expressed in any cell, e.g., an immune effector cell (e.g., a T cell or NK cell) as described herein.

The term "signaling domain" refers to a functional part of a protein that functions by transmitting information inside a cell: cellular activity is regulated by means of defined signaling pathways, either by the production of second messengers or by functioning as effectors in response to such messengers.

As used herein, the term "CD 33" refers to a differentiation epitope 33 protein that is an antigenic determinant detectable on leukemia cells as well as on myeloid-lineage normal precursor cells. Human and murine amino acid sequences and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD33 can be found as UniProt/Swiss-Prot accession number P20138, and the nucleotide sequence encoding human CD33 can be found as accession number NM — 001772.3. In one aspect, the antigen binding portion of the CAR recognizes and binds to an epitope within the extracellular domain of the CD33 protein or fragment thereof. In one aspect, the CD33 protein is expressed on cancer cells. As used herein, "CD 33" includes proteins comprising mutations (e.g., point mutations), fragments, insertions, deletions, and splice variants of full-length wild-type CD 33.

As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins and may be derived from natural sources or from recombinant sources. The antibody may be a tetramer of immunoglobulin molecules.

The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and refers to an antigen-binding domain, e.g., an antigenic determinant variable region of an intact antibody, sufficient to cause recognition and specific binding of the antibody fragment to a target (e.g., an antigen). Examples of antibody fragments include, but are not limited to, Fab ', F (ab')₂And Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdabs (VL or VH), camelid (camelid) VHH domains, and multispecific molecules formed from antibody fragments, such as bivalent fragments comprising two or more (e.g., two) Fab fragments linked by a disulfide bond at the hinge region, or two or more (e.g., two) isolated CDRs or other epitope-binding fragments of a linked antibody. Antibody fragments can also be incorporated into single domain antibodies, large antibodies (maxibodies), minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Holli @) Ringer and Hudson,2005, Nature Biotechnology,23: 1126: 1136, 2005). Antibody fragments can also be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. patent No. 6,703,199, which describes fibronectin polypeptide minibodies).

The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are consecutively linked by means of a flexible short polypeptide linker and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise indicated, as used herein, a scFv can have VL variable regions and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of a polypeptide), can comprise a VL-linker-VH, or can comprise a VH-linker-VL.

As used herein, the term "complementarity determining region" or "CDR" refers to an amino acid sequence that confers antigen specificity and binding affinity within the variable region of an antibody. For example, there are three CDRs (HCDR1, HCDR2, and HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, and LCDR3) in each light chain variable region. The precise amino acid sequence boundaries of a given CDR can be determined using any of a variety of well-known protocols, including those defined by Kabat et al (1991), "Sequences of Proteins of Immunological Interest", 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et Al, (1997) JMB 273,927-948 ("Chothia" numbering scheme); or a combination thereof. According to the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35(HCDR1), 50-65(HCDR2), and 95-102(HCDR 3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34(LCDR1), 50-56(LCDR2) and 89-97(LCDR 3). According to the Chothia numbering scheme, in some embodiments, the CDR amino acids in the VH are numbered 26-32(HCDR1), 52-56(HCDR2), and 95-102(HCDR 3); and the CDR amino acid residues in the VL are numbered 26-32(LCDR1), 50-52(LCDR2) and 91-96(LCDR 3). In the combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both. For example, in some embodiments, the CDRs correspond to amino acid residues 26-35(HCDR1), 50-65(HCDR2) and 99-102(HCDR3) in a VH (e.g., a mammalian VH, e.g., a human VH) and amino acid residues 24-34(LCDR1), 50-56(LCDR2) and 89-97(LCDR3) in a VL (e.g., a mammalian VL, e.g., a human VL).

The portion of the CAR composition of the invention comprising an antibody or antibody fragment thereof may, for example, exist in a variety of forms wherein the antigen binding domain is represented as part of a polypeptide chain, including, for example, single domain antibody fragments (sdabs), single chain Antibodies (scFv), or, for example, humanized or human Antibodies (Harlow et al, 1999, cited in: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, cited in Antibodies: A Laboratory, Cold Spring Harbor, New York, Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Bird et al, 1988, Science 242: 423-. In one aspect, the antigen binding domain of the CAR composition of the invention comprises an antibody fragment. In yet another aspect, the CAR comprises an antibody fragment comprising an scFv.

As used herein, the term "binding domain" or "antibody molecule" refers to a protein comprising at least one immunoglobulin variable domain sequence, e.g., an immunoglobulin chain or fragment thereof. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In one embodiment, the antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences has binding specificity for a second epitope. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies are specific for no more than two antigens. Bispecific antibody molecules are characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope.

The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation, which normally determines the class to which an antibody belongs.

The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

The term "recombinant antibody" refers to an antibody produced using recombinant DNA techniques, e.g., an antibody expressed by a phage or yeast expression system. The term should also be construed to mean an antibody that has been produced by synthesizing a DNA molecule encoding the antibody and expressing the antibody protein or by synthesizing the amino acid sequence of a particular antibody, where the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence techniques available and well known in the art.

The term "antigen" or "Ag" refers to a molecule that elicits an immune response. Such an immune response may involve antibody production, or activation of specific immunocompetent cells, or both. The skilled person will appreciate that any macromolecule, including virtually all proteins or peptides, can act as an antigen. In addition, the antigen may be derived from recombinant DNA or genomic DNA. The skilled person will understand that any DNA comprising a nucleotide sequence or part of a nucleotide sequence encoding a protein which elicits an immune response therefore encodes an "antigen" as that term is used herein. In addition, one skilled in the art will appreciate that an antigen need not be encoded entirely by the full-length nucleotide sequence of a gene. It will be readily apparent that the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. In addition, the skilled person will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that the antigen may be produced synthetically or may be derived from a biological sample, or may be a macromolecule in addition to a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or fluids with other biological components.

The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means, including, but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, a reduction in tumor cell proliferation, a reduction in tumor cell survival, or an improvement in a variety of physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be demonstrated by the ability of the peptides, polynucleotides, cells and antibodies of the invention to prevent the appearance of tumors at the primary site.

The term "anti-cancer effect" refers to a biological effect that can be exhibited by a variety of means, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in a variety of physiological symptoms associated with a cancer condition. An "anti-cancer effect" can also be demonstrated by the ability of peptides, polynucleotides, cells and antibodies to prevent the appearance of cancer at the primary site. The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means, including, but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

The term "autologous" refers to any substance that is derived from the same individual into whom the substance is later to be reintroduced into the individual.

The term "allogeneic" refers to any substance derived from a different animal of the same species as the individual into which the substance is introduced. When the genes at one or more loci are not identical, two or more individuals are said to be allogeneic with respect to each other. In some aspects, allogeneic substances from individuals of the same species may be sufficiently genetically dissimilar to interact antigenically.

The term "xenogeneic" refers to grafts derived from animals of different species.

The term "apheresis" as used herein refers to art-recognized extracorporeal methods by which blood of a donor or patient is removed from the donor or patient and passed through a device that separates selected specific components and returns the remainder to the circulation of the donor or patient, e.g., by re-transfusion. Thus, in the context of "apheresis" refers to a sample obtained using apheresis.

The term "combination" refers to a fixed combination or combined administration in one dosage unit form, wherein a compound of the invention and a combination partner (e.g. another drug as explained below, also referred to as "therapeutic agent" or "adjuvant") may be administered independently at the same time or separately within respective time intervals, in particular wherein these time intervals allow the combination partner to show a cooperative, e.g. synergistic effect. The individual components may be packaged in kits or separately. One or both of the components (e.g., powder or liquid) may be reconstituted or diluted to the desired dosage prior to administration. The terms "co-administration" or "combined administration" and the like as used herein are intended to encompass the administration of a selected combination partner to a single subject (e.g., patient) in need thereof, and are intended to include treatment regimens in which the drugs are not necessarily administered by the same route of administration or simultaneously. The term "pharmaceutical combination" as used herein means a product resulting from mixing or combining more than one active ingredient and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients, e.g. the compounds of the invention and the combination partners, are both administered to the patient simultaneously in a single entity or dose. The term "non-fixed combination" means that the active ingredients, e.g. a compound of the invention and a combination partner, are both administered to a patient as separate entities simultaneously, simultaneously or sequentially with no specific time constraints, wherein such administration provides therapeutically effective levels of both compounds within the body of the patient. The latter is also suitable for cocktail therapy, e.g. administration of three or more active ingredients.

The term "cancer" refers to a disease characterized by rapid and uncontrolled growth of abnormal cells. Cancer cells can spread to other parts of the body locally or through the blood stream and lymphatic system. Examples of various cancers are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid tumors and liquid tumors, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes premalignant as well as malignant cancers and tumors.

"derived from … …" as that term is used herein refers to the relationship between a first molecule and a second molecule. It generally refers to the structural similarity between a first molecule and a second molecule and does not imply or include process or source limitations on the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from the CD3 ζ molecule, the intracellular signaling domain retains sufficient CD3 ζ structure to have the desired function, i.e., the ability to generate a signal under suitable conditions. It does not imply or include a limitation to the specific process of generating an intracellular signaling domain, for example, it does not mean that, in order to provide an intracellular signaling domain, one must start with the CD3 ζ sequence and delete an unwanted sequence, or confer a mutation to achieve an intracellular signaling domain.

The phrase "a disease associated with expression of CD 33" as used herein includes, but is not limited to, a disease associated with cells expressing CD33 (e.g., wild-type or mutant CD33) or a condition associated with cells expressing or expressing CD33 (e.g., wild-type or mutant CD33), including, for example, a proliferative disease, such as a cancer or malignancy, or a precancerous disease, such as myelodysplasia, myelodysplastic syndrome, or pre-leukemia; or a non-cancer related indication associated with cells expressing CD33 (e.g., wild-type or mutant CD 33). For the avoidance of doubt, a disease associated with expression of CD33 may include a condition associated with cells that do not currently express CD33, e.g., because CD33 expression has been down-regulated, e.g., because of treatment with a molecule that targets CD33 (e.g., a CD33 inhibitor as described herein), but that have historically expressed CD 33. In one aspect, the cancer associated with expression of CD33 (e.g., wild-type or mutant CD33) is a hematologic cancer. In one aspect, hematological cancers include, but are not limited to, Acute Myeloid Leukemia (AML), myelodysplastic and myelodysplastic syndromes, myelofibrosis and myeloproliferative tumors, Acute Lymphoid Leukemia (ALL), hairy cell leukemia, prolymphocytic leukemia, Chronic Myeloid Leukemia (CML), blastic plasma-like dendritic cell tumors, and the like. Diseases associated with expression of CD33 (e.g., wild-type or mutant CD33) include, but are not limited to, for example, atypical and/or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with expression of CD33 (e.g., wild-type or mutant CD 33). Non-cancer related indications associated with CD33 expression (e.g., wild-type or mutant CD33) may also be included. In embodiments, non-cancer related indications associated with expression of CD33 include, but are not limited to, for example, autoimmune diseases (e.g., lupus), inflammatory diseases (allergy and asthma), and transplantation. In some embodiments, the cells expressing the tumor antigen express or at any time overexpress mRNA encoding the tumor antigen. In one embodiment, the tumor antigen expressing cell produces a tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal levels or at reduced levels. In one embodiment, a cell expressing a tumor antigen produces detectable levels of tumor antigen protein at one time point and subsequently produces substantially no detectable tumor antigen protein.

The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies or antibody fragments of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, one or more amino acid residues internal to the CAR of the invention can be replaced with other amino acid residues from the same side chain family, and the altered CAR can be tested using the functional assays described herein.

The term "stimulation" refers to a primary response induced by the binding of a stimulating molecule (e.g., the TCR/CD3 complex) to its corresponding ligand, which primary response thereby mediates a signaling event, such as, but not limited to, signaling via the TCR/CD3 complex. Stimulation may mediate altered expression of certain molecules, such as by downregulating reorganization of TGF- β and/or cytoskeletal structure, among others.

The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that modulates primary activation of the TCR complex in a stimulatory manner in at least some aspect of the T cell signaling pathway. In one aspect, the primary signal is initiated, e.g., by binding of the TCR/CD3 complex to peptide-loaded MHC molecules and results in mediating T cell responses including, but not limited to, proliferation, activation, differentiation, and the like. The primary cytoplasmic signaling sequence (also referred to as the "primary signaling domain") that functions in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of primary cytoplasmic signaling sequences containing ITAMs that are particularly useful in the present invention include, but are not limited to, those derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ∈, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), fceri, and CD66d, DAP10, and DAP 12. In a particular CAR of the invention, the intracellular signaling domain in any one or more of the CARs of the invention comprises an intracellular signaling sequence, e.g., the primary signaling sequence of CD 3-zeta. In a particular CAR of the invention, the primary signaling sequence of CD 3-zeta is the sequence provided as SEQ ID NO:9 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In a particular CAR of the invention, the primary signaling sequence of CD 3-zeta is the sequence provided in SEQ ID NO:10 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

The term "antigen presenting cell" or "APC" refers to an immune system cell such as an accessory cell (e.g., B cell, dendritic cell, etc.) that presents a foreign antigen complexed with a Major Histocompatibility Complex (MHC) on its surface. T cells can recognize these complexes using their T Cell Receptor (TCR). The APC processes antigens and presents them to T cells.

As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. In embodiments, the intracellular signaling domain transduces effector function signals and directs the cell to perform a specialized function. Although complete intracellular signaling domains can be used, in many cases the use of complete strands is not required. Truncated portions of the intracellular signaling domain are used to the extent that such truncated portions can be used in place of the entire chain, so long as it conducts effector function signals. The term "intracellular signaling domain" is therefore intended to include any truncated portion of an intracellular signaling domain sufficient to signal effector function. The intracellular signaling domain can generate a signal that promotes immune effector function of a CAR-containing cell (e.g., a CART cell or a CAR-expressing NK cell). Examples of immune effector functions (e.g., in CART cells or CAR-expressing NK cells) include cytolytic and helper activities, including secretion of cytokines.

In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules that respond to primary stimuli or antigen-dependent stimuli. In one embodiment, the intracellular signaling domain may comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules that respond to costimulatory signals or antigen-independent stimuli. For example, in the case of an immune effector cell expressing a CAR (e.g., a CART cell or a CAR-expressing NK cell), the primary intracellular signaling domain may comprise a cytoplasmic sequence of a T cell receptor and the costimulatory intracellular signaling domain may comprise a cytoplasmic sequence from a co-receptor or a co-stimulatory molecule.

The primary intracellular signaling domain may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3 ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), fcε RI and CD66d, DAP10, and DAP 12.

The term "zeta" or alternatively "zeta chain", "CD 3-zeta" or "TCR-zeta" is defined as the equivalent residue of a protein provided as GenBan accession number BAG36664.1 or from a non-human species (e.g., mouse, rodent, monkey, ape, etc.), and the "zeta stimulating domain" or alternatively "CD 3-zeta stimulating domain" or "TCR-zeta stimulating domain" is defined as the amino acid residue from the zeta chain cytoplasmic domain sufficient to functionally transmit the initial signal necessary for T cell activation. In one aspect, the cytoplasmic domain of ζ comprises residues 52 through 164 of GenBank accession No. BAG36664.1 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) as a functional ortholog thereof. In one aspect, the "zeta stimulating domain" or "CD 3-zeta stimulating domain" is the sequence provided as SEQ ID NO 9. In one aspect, the "zeta stimulating domain" or "CD 3-zeta stimulating domain" is the sequence provided as SEQ ID NO 10.

The term "co-stimulatory molecule" refers to a corresponding binding partner on a T cell that specifically binds to a co-stimulatory ligand, and thus mediates a co-stimulatory response (such as, but not limited to, proliferation) by the T cell. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for a high immune response. Costimulatory molecules include, but are not limited to: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signal molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1(CD11a/CD a), 4-1BB (CD137), B a-H a, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHT TR), KIRDS a, SLAMF a, NKp a (KLRF a), NKp a, CD a alpha, CD a beta, IL2 a gamma, ITIL 7 alpha, IT3672, VLITGA 72, VLITGA a, GAITGB 72, GAITGA a, GAITGB 11-a, GAITGA a, GAITGB 11, GAITGA a, GAITGB a, GAIT11, CD a, GAITGA a, GAITGB 72, GAITGA a, CD a, GAITGB 11-a, GAITGB 11, GAITGA a, GAITGB a, CD a, GAITGA a, GAITGB 11-a, GAITCD a, GAITGA a, CD a, GAITGB 11-a, CD a, GAITGB 11, SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds to CD 83.

The costimulatory intracellular signaling domain refers to the intracellular portion of the costimulatory molecule.

The intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived or the entire native intracellular signaling domain or a functional fragment thereof.

The term "4-1 BB" refers to a TNFR superfamily member having an amino acid sequence provided as GenBank accession No. AAA62478.2 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.); and the "4-1 BB co-stimulatory domain" is defined as amino acid residue 214-255 of GenBank accession AAA62478.2 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In one aspect, the "4-1 BB co-stimulatory domain" is the sequence provided as SEQ ID NO:7 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

As the term is used herein, the term "immune effector cell" refers to a cell involved in an immune response, e.g., involved in promoting an immune effector response. Examples of immune effector cells include T cells, e.g., α/β T cells and γ/δ T cells, B cells, Natural Killer (NK) cells, natural killer T (nkt) cells, mast cells, and myeloid cell-derived phagocytic cells.

As the term is used herein, "immune effector function or immune effector response" refers to, for example, the enhancement of immune effector cells or the function or response that promotes immune attack on target cells. For example, immune effector function or response refers to the characteristics of T cells or NK cells that promote killing of target cells or inhibit growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including secretion of cytokines.

The term "encoding" refers to the intrinsic properties of, and the biological properties resulting from, a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, that serves as a template in biological processes to synthesize other polymers and macromolecules with defined nucleotide sequences (e.g., rRNA, tRNA, and mRNA) or defined amino acid sequences. Thus, a gene, cDNA or RNA encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and which is usually provided in the sequence listing, and the non-coding strand used as a template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of the gene or cDNA.

Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence" encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may contain introns in some form.

The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material or composition that is effective to achieve a particular biological result as described herein.

The term "endogenous" refers to any substance that is derived from or produced within an organism, cell, tissue, or system.

The term "exogenous" refers to any substance introduced from or produced outside of an organism, cell, tissue, or system.

The term "expression" refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

The term "transfer vector" refers to a composition of matter that comprises an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes autonomously replicating plasmids or viruses. The term should also be construed to further include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, e.g., polylysine compounds, liposomes, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

The term "lentivirus" refers to a genus of the family Retroviridae (Retroviridae). Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information to the DNA of host cells, and thus they are one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are examples of lentiviruses.

The term "lentiviral vector" refers to a vector derived from at least a portion of the lentiviral genome, particularlyIt includes self-inactivating lentiviral vectors as provided in Milone et al, mol. ther.17(8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, for example, but are not limited to, those from Oxford BioMedica

Gene delivery technology, LENTIMAX from Lentigen^TMVector systems, and the like. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.

The term "homologous" or "identity" refers to a subunit sequence identity between two polymer molecules (e.g., between two nucleic acid molecules (e.g., between two DNA molecules or two RNA molecules) or between two polypeptide molecules). When a subunit position in both molecules is occupied by the same monomeric subunit, e.g., if a position in each of the two DNA molecules is occupied by adenine, then they are homologous or identical at that position. Identity between two sequences varies directly with the number of matching or homologous positions, e.g., if half the positions (e.g., 5 positions in a polymer of 10 subunits in length) in two sequences are homologous, then the two sequences are 50% homologous; the two sequences are 90% homologous if 90% of the positions (e.g., 9 out of 10 positions) are matching or homologous.

"humanized" forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab ', F (ab's) of an antibody) that contain minimal sequence derived from non-human immunoglobulin ₂Or other antigen binding subsequences). For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues in a Complementarity Determining Region (CDR) from the recipient are replaced by residues in a CDR of the desired specificity, affinity and capacity from a non-human species (donor antibody) such as mouse, rat or rabbit. In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies/antibody fragments may compriseResidues that are not present in either the recipient antibody or the imported CDR or framework sequences. These modifications can further modify and optimize antibody or antibody fragment performance. Typically, a humanized antibody or antibody fragment thereof comprises substantially all of at least 1, and typically 2, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a substantial portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al, Nature,321:522-525, 1986; reichmann et al, Nature,332: 323-E329, 1988; presta, curr, Op, struct, biol.,2: 593-.

"fully human (immunoglobulin)" refers to an immunoglobulin, such as an antibody or antibody fragment, in which the entire molecule is of human origin or consists of the same amino acid sequence as the antibody or human form of the immunoglobulin.

The term "isolated" means altered or removed from the native state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide, partially or completely separated from the coexisting materials of its natural state, is "isolated. An isolated nucleic acid or protein may be present in a substantially purified form, or may be present in a non-natural environment (e.g., a host cell).

In the context of the present invention, the following abbreviations are used for the common nucleic acid bases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine and "U" refers to uridine.

The term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be contiguous with each other and, for example, in the case where it is desired to join two protein coding regions, in the same open reading frame.

The term "parenteral" administration of the immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, intratumoral, or infusion techniques.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single-stranded or double-stranded form. Unless specifically limited, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also inherently includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081, (1991); Ohtsuka et al, J.biol.chem.260: 2605-.

The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids and does not limit the maximum number of amino acids that can make up the sequence of the protein or peptide. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and also to longer chains, which are commonly referred to in the art as proteins, which exist in many types. "polypeptide" includes, for example, biologically active fragments of polypeptides, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide includes a native peptide, a recombinant peptide, or a combination thereof.

The term "promoter" refers to a DNA sequence recognized by a cellular synthesis apparatus or an introduced synthesis apparatus that is required to initiate sequence-specific transcription of a polynucleotide.

The term "promoter/regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter/regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue-specific manner.

The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all of the physiological conditions of the cell.

The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

The term "tissue-specific" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoded or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is the cell of the tissue type to which the promoter corresponds.

The term "cancer-associated antigen" or "tumor antigen" refers interchangeably to a molecule (typically a protein, sugar, or lipid) that is expressed intact or as a fragment (e.g., MHC/peptide) on the surface of a cancer cell and can be used to preferentially direct an agent to the cancer cell. In some embodiments, the tumor antigen is a marker expressed by both normal and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, the tumor antigen is a cell surface molecule that is overexpressed in cancer cells compared to normal cells (e.g., 1-fold overexpressed, 2-fold overexpressed, 3-fold or more overexpressed compared to normal cells). In some embodiments, the tumor antigen is a cell surface molecule that is not amenable to synthesis in cancer cells, e.g., a molecule that contains deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, the tumor antigen will be expressed exclusively, either intact or as a fragment (e.g., MHC/peptide), on the surface of cancer cells and not synthesized or expressed on the surface of normal cells. In some embodiments, the CARs of the invention include CARs that comprise an antigen binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Typically, peptides derived from endogenous proteins fill the pits of Major Histocompatibility Complex (MHC) class I molecules and are recognized by T Cell Receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and/or tumor-specific peptide/MHC complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting peptides derived from viral or tumor antigens in the context of Human Leukocyte Antigen (HLA) -A1 or HLA-A2 have been described (see, e.g., Sastry et al, J Virol.201185 (5): 1935-. For example, TCR-like antibodies can be identified from screening libraries, such as human scFv phage display libraries.

The term "tumor-supporting antigen" or "cancer-supporting antigen" interchangeably refers to a molecule (typically a protein, sugar, or lipid) that is expressed on the surface of a cell that is itself non-cancerous, but supports the cancer cell, for example, by promoting growth or survival of the cancer cell (e.g., against an immune cell). Exemplary cells of this type include mesenchymal cells and Myeloid Derived Suppressor Cells (MDSCs). The tumor supporting antigen itself need not be functional in supporting tumor cells, so long as the antigen is present on the cells that support the cancer cells.

The term "flexible polypeptide linker" or "linker" as used in the context of an scFv refers to a peptide linker consisting of amino acid, such as glycine and/or serine residues, wherein the residues are used alone or in combination to link together the variable heavy and variable light chain regions. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser) n (SEQ ID NO:38), wherein n is a positive integer equal to or greater than 1. For example, n is 1, n is 2, n is 3, n is 4, n is 5 and n is 6, n is 7, n is 8, n is 9 and n is 10. In one embodiment, the flexible polypeptide linker includes, but is not limited to, (Gly4Ser)4(SEQ ID NO:27) or (Gly4Ser)3(SEQ ID NO: 28). In another embodiment, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 29). Also included within the scope of the present invention are linkers as described in WO 2012/138475, which are incorporated herein by reference.

As used herein, a 5' cap (also referred to as an RNA cap, RNA 7-methylguanosine cap or RNA m)⁷G cap) is a modified guanine nucleotide added to the "front end" or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group linked to the first transcribed nucleotide. Its presence is critical for ribosome recognition and protection from rnase action. The cap addition is coupled to transcription and occurs in a co-transcriptional manner, each affecting the other. Shortly after transcription begins, the 5' end of the mRNA being synthesized is bound by a cap synthesis complex that binds to RNA polymerase. This enzymatic complex catalyzes the chemical reaction required for mRNA capping. The synthetic process advances as a multi-step biochemical reaction. The capping moiety may be modified to modulate a function of the mRNA, such as its stability or translation efficiency.

As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, that has been synthesized in vitro. Typically, the in vitro transcribed RNA is produced from an in vitro transcription vector. The in vitro transcription vector contains a template for generating in vitro transcribed RNA.

As used herein, "polya" is a series of adenosines linked to mRNA by polyadenylation. In a preferred embodiment of the transient expression construct the number of polyadenylation is between 50 and 5000 (SEQ ID NO:30), preferably more than 64, more preferably more than 100, most preferably more than 300 or 400. The polyadenylation sequence may be chemically or enzymatically modified to modulate mRNA function such as localization, stability or translation efficiency.

As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylacyl moiety, or modified variant thereof, to a messenger RNA molecule. In eukaryotes, most messenger rna (mrna) molecules are polyadenylated at the 3' end. The 3' poly a tail is a long adenine nucleotide sequence (often several hundred) that is added to the pre-mRNA by the action of an enzyme (poly a polymerase). In higher eukaryotes, a poly A tail is added to the transcript containing the specific sequence (polyadenylation signal). The poly a tail and the protein to which it is bound help protect the mRNA from exonuclease degradation. Polyadenylation is also important for transcription termination, mRNA export from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but may additionally occur later in the cytoplasm. After transcription has been terminated, the mRNA strand is cleaved by the action of an endonuclease complex that binds to RNA polymerase. The cleavage site is generally characterized by the presence of the base sequence AAUAAA in the vicinity of the cleavage site. After the mRNA has been sheared, an adenosine residue is added to the free 3' end at the site of the shear.

As used herein, "transient" refers to the expression of a non-integrated transgene over a period of hours, days, or weeks, wherein the period of expression is less than the period of expression of the gene if it is integrated into the genome or contained within a stable plasmid replicon in the host cell.

As used herein, the terms "treat," "treatment," and "treating" refer to reducing or lessening the progression, severity, and/or duration of a proliferative disease or ameliorating one or more symptoms (preferably, one or more perceptible symptoms) of a proliferative disease as a result of administering one or more therapies (e.g., one or more therapeutic agents, such as a CAR of the invention). In particular embodiments, the terms "treat," "treatment," and "treating" refer to ameliorating at least one measurable physical parameter of a proliferative disease, such as tumor growth, that is not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to inhibiting the progression of a proliferative disease, either physically (e.g., by stabilizing a perceptible symptom), physiologically (e.g., by stabilizing a physical parameter), or both. In other embodiments, the terms "treat," "therapy," and "treating" refer to reducing or stabilizing tumor size or cancer cell count.

The term "signal transduction pathway" refers to the biochemical relationship between a variety of signal transduction molecules that function in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and molecular complexes capable of receiving and transmitting signals across cell membranes.

The term "subject" is intended to include living organisms (e.g., mammals, humans) in which an immune response can be elicited.

The term "substantially purified" cell refers to a cell that is substantially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they normally associate in their naturally occurring state. In some cases, a substantially purified cell population refers to a homogeneous cell population. In other cases, this term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cell is not cultured in vitro.

The term "therapeutic" as used herein means treatment. The therapeutic effect is achieved by achieving a reduction, inhibition, alleviation or eradication of the disease state.

The term "prevention" as used herein means the prophylactic or protective treatment of a disease or condition.

In the context of the present invention, "tumor antigen" or "antigen of a hyperproliferative disease" or "antigen associated with a hyperproliferative disease" refers to an antigen that is common to the particular hyperproliferative disease. In certain aspects, the hyperproliferative disease antigens of the present invention are derived from cancers including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, renal cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.

The term "transfection" or "transformation" or "transduction" refers to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. Cells include the primary subject cell and progeny thereof.

The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a corresponding binding partner (e.g., a stimulatory molecule and/or co-stimulatory molecule present on a T cell) protein present in a sample, but which does not substantially recognize or bind to other molecules in the sample.

As used herein, "Regulatable Chimeric Antigen Receptor (RCAR)" refers to a group of polypeptides, generally two polypeptides in the simplest embodiment, that, when in an immune effector cell, provide specificity for the target cell (typically a cancer cell) and provide intracellular signal generation for the cell. In some embodiments, the RCAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and/or a co-stimulatory molecule as defined herein in the context of a CAR molecule. In some embodiments, the set of polypeptides in the RCAR are not contiguous with each other, e.g., are in different polypeptide chains. In some embodiments, the RCAR includes a dimerization switch, wherein the dimerization switch can couple polypeptides to each other, e.g., can couple an antigen binding domain to an intracellular signaling domain, in the presence of a dimerization molecule. In some embodiments, the RCAR is expressed in a cell (e.g., an immune effector cell) as described herein (e.g., a cell expressing RCAR (also referred to herein as an "RCARX cell"). in one embodiment, the RCARX cell is a T cell and is referred to as an RCART cell.

As the term is used herein, "membrane anchor" or "membrane-binding domain" refers to a polypeptide or moiety, e.g., myristoyl, sufficient to anchor an extracellular or intracellular domain to the plasma membrane.

As used herein, such as in reference to RCAR, a "switch domain" refers to an entity, typically a polypeptide-based entity, that associates with another switch domain in the presence of a dimerizing molecule. Association results in functional coupling of a first entity linked (e.g., fused) to a first switch domain and a second entity linked (e.g., fused) to a second switch domain. The first and second switch domains are collectively referred to as a dimerization switch. In embodiments, the first and second switch domains are identical to each other, e.g., they are polypeptides having the same primary amino acid sequence and are collectively referred to as a homodimerization switch. In embodiments, the first and second switch domains are different from each other, e.g., they are polypeptides having different primary amino acid sequences and are collectively referred to as heterodimerization switches. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is a polypeptide-based (e.g., FKBP or FRB-based) entity, and the dimerizing molecule is a small molecule, e.g., a rapamycin analog (rapalogue). In embodiments, the switch domain is a polypeptide-based entity, e.g., an scFv that binds a myc peptide, and the dimerizing molecule is a polypeptide, a fragment thereof, or a polypeptide multimer, e.g., a myc ligand or a myc ligand multimer that binds to one or more myc scfvs. In embodiments, the switch domain is a polypeptide-based entity, e.g., a myc receptor, and the dimerizing molecule is an antibody or fragment thereof, e.g., a myc antibody.

As the term is used herein, for example in reference to RCAR, a "dimerizing molecule" refers to an entity that facilitates association of a first switch domain with a second switch domain. In embodiments, the dimerizing molecule does not occur naturally in the subject or at a concentration that would result in significant dimerization. In embodiments, the dimerizing molecule is a small molecule, e.g., rapamycin or a rapamycin analog, e.g., RAD 001.

The term "bioequivalent" refers to an amount of a drug other than a reference compound (e.g., RAD001) that is required to produce an effect equivalent to that produced by a reference dose or reference amount of the reference compound (e.g., RAD 001). In one embodiment, the effect is mTOR inhibition level, e.g., as measured by P70S6 kinase inhibition, e.g., as assessed in an in vivo or in vitro assay, e.g., as measured by an assay described herein (e.g., Boulay assay), or measuring phosphorylation S6 level by western blot. In one embodiment, the effect is a change in the ratio of PD-1 positive/PD-1 negative immune effector cells (e.g., T cells or NK cells) as measured by cell sorting. In one embodiment, the bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of inhibition of P70S6 kinase as a reference dose or reference amount of a reference compound. In one embodiment, the bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of change in the ratio of PD-1 positive/PD-1 negative immune effector cells (e.g., T cells or NK cells) as a reference dose or reference amount of a reference compound.

The term "immunopotentiating low dose" when used in connection with an mTOR inhibitor (e.g., an allosteric mTOR inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR inhibitor) refers to a dose of an mTOR inhibitor that partially, but not completely, inhibits mTOR activity (e.g., as measured by inhibition of P70S6 kinase activity). Methods for assessing mTOR activity (e.g., by inhibiting P70S6 kinase) are discussed herein. This dose is not sufficient to result in complete immunosuppression, but is sufficient to enhance the immune response. In one embodiment, the immunopotentiating low dose of the mTOR inhibitor results in a decrease in the number of PD-1 positive immune effector cells (e.g., T cells or NK cells) and/or an increase in the number of PD-1 negative immune effector cells (e.g., T cells or NK cells), or an increase in the ratio of PD-1 negative immune effector cells (e.g., T cells or NK cells)/PD-1 positive immune effector cells (e.g., T cells or NK cells).

In one embodiment, an immunopotentiating low dose of an mTOR inhibitor results in an increase in the number of naive T cells. In one embodiment, an immunopotentiating low dose of an mTOR inhibitor results in one or more of the following:

increased expression of one or more of the following markers: for example, CD62L on memory T cells (e.g., memory T cell precursors) ^{Height of}、CD127^{Height of}、CD27⁺And BCL 2;

e.g., decreased expression of KLRG1 on memory T cells (e.g., memory T cell precursors); and

an increase in the number of memory T cell precursors, such as cells having any one or a combination of the following characteristics: CD62L^{Height of}Increase, CD127^{Height of}Increase, CD27⁺Increase, decrease KLRG1, and increase BCL 2;

wherein, for example, any of the changes described above occur, e.g., at least temporarily, as compared to an untreated subject.

"refractory" as used herein, refers to a disease that does not respond to treatment, e.g., cancer. In embodiments, a refractory cancer may be resistant to treatment prior to or at the beginning of treatment. In other embodiments, refractory cancer may become resistant during treatment. Refractory cancers are also referred to as drug resistant cancers.

As used herein, "recurrent" or "recurrence" refers to the recurrence of signs and symptoms of a disease (e.g., cancer) or disease (e.g., cancer) after an amelioration or response phase, e.g., after a previous treatment of a therapy (e.g., cancer therapy). For example, the reactive stage may involve a cancer cell level falling below a certain threshold, e.g., below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Reoccurrence may involve an increase in cancer cell levels above a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%.

The range is as follows: throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a limitation on the scope of the present invention. Accordingly, the description of a range should be considered to have all of the possible subranges specifically disclosed as well as individual numerical values within that range. For example, a range such as that described from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, and the like, as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range (e.g., 95-99% identity) includes a range having 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the width of the range.

Detailed Description

Provided herein are compositions of matter and methods of use for treating or preventing diseases, such as cancer, using a CD33 Chimeric Antigen Receptor (CAR).

In one aspect, the invention provides a plurality of Chimeric Antigen Receptors (CARs) comprising an antibody or antibody fragment engineered to specifically bind to a CD33 protein or fragment thereof. In one aspect, the invention provides a cell (e.g., an immune effector cell, e.g., a T cell or NK cell) engineered to express a CAR, wherein the cell (e.g., "CART") exhibits anti-tumor properties. In one aspect, the cell is transformed with a CAR and the CAR or at least a portion of the CAR is expressed on the surface of the cell. In some embodiments, a cell (e.g., an immune effector cell, e.g., a T cell or NK cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell can stably express the CAR. In another embodiment, the cell (e.g., an immune effector cell, e.g., a T cell or NK cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding the CAR. In some such embodiments, the cell can transiently express the CAR.

In one aspect, the CD33 binding domain (e.g., a human or humanized CD33 binding domain) of the CAR is an scFv antibody fragment. In one aspect, such antibody fragments are functional in that they retain equivalent binding affinity, e.g., they bind the same antigen with comparable efficacy, e.g., they are IgG antibodies having the same heavy chain variable region and light chain variable region. In one aspect, such antibody fragments are functional in that they provide a biological response that may include, but is not limited to, activating an immune response, inhibiting signal transduction from its target antigen, inhibiting kinase activity, and the like, as will be understood by the skilled artisan.

In some aspects, the antibodies of the invention are incorporated into a Chimeric Antigen Receptor (CAR). In one aspect, the CAR comprises a polypeptide sequence provided herein as SEQ ID NOS: 48-56.

In one aspect, the CD 33-binding domain (e.g., humanized or human CD 33-binding domain) portion of the CAR of the invention is encoded by a transgene whose sequence has been codon optimized for expression in mammalian cells. In one aspect, the complete CAR construct of the invention is encoded by a transgene whose entire sequence has been codon optimized for expression in mammalian cells. Codon optimization refers to the following findings: the frequency of occurrence of synonymous codons (i.e., codons encoding the same amino acid) in the coding DNA is biased among different species. This codon degeneracy allows the same polypeptide to be encoded by multiple nucleotide sequences. Various codon optimization methods are known in the art and include, for example, at least the methods disclosed in U.S. Pat. nos. 5,786,464 and 6,114,148.

In one aspect, the human CD33 binding domain comprises the scFv portion provided in SEQ ID NOS: 39-47. In one aspect, the human CD33 binding domain comprises the scFv portion provided in SEQ ID NO: 39. In one aspect, the human CD33 binding domain comprises the scFv portion provided in SEQ ID NO: 40. In one aspect, the human CD33 binding domain comprises the scFv portion provided in SEQ ID NO 41. In one aspect, the human CD33 binding domain comprises the scFv portion provided in SEQ ID NO: 42. In one aspect, the human CD33 binding domain comprises the scFv portion provided in SEQ ID NO 43. In one aspect, the human CD33 binding domain comprises the scFv portion provided in SEQ ID NO: 44. In one aspect, the human CD33 binding domain comprises the scFv portion provided in SEQ ID NO: 45. In one aspect, the human CD33 binding domain comprises the scFv portion provided in SEQ ID NO. 46. In one aspect, the human CD33 binding domain comprises the scFv portion provided in SEQ ID NO: 47. In one aspect, the CAR of the invention combines the antigen binding domain of a specific antibody with an intracellular signaling molecule. For example, in some aspects, intracellular signaling molecules include, but are not limited to, the CD 3-zeta chain, the 4-1BB, and the CD28 signaling modules, and combinations thereof. In one aspect, the antigen binding domain binds to CD 33. In one aspect, the CD33CAR comprises a CAR selected from the sequences provided in one or more of SEQ ID NOs 48-56. In one aspect, the CD33CAR comprises the sequence provided in SEQ ID No. 48. In one aspect, the CD33CAR comprises the sequence provided in SEQ ID No. 49. In one aspect, the CD33CAR comprises the sequence provided in SEQ ID NO: 50. In one aspect, the CD33CAR comprises the sequence provided in SEQ ID NO: 51. In one aspect, the CD33CAR comprises the sequence provided in SEQ ID No. 52. In one aspect, the CD33CAR comprises the sequence provided in SEQ ID No. 53. In one aspect, the CD33CAR comprises the sequence provided in SEQ ID No. 54. In one aspect, the CD33CAR comprises the sequence provided in SEQ ID NO: 55. In one aspect, the CD33CAR comprises the sequence provided in SEQ ID NO: 56.

In addition, the present invention provides CD33 CAR compositions and their use in medicaments or methods for treating cancer or any malignancy or autoimmune disease, among other diseases involving cells or tissues expressing CD 33.

In one aspect, the CARs of the invention can be used to eradicate normal cells expressing CD33 and are thus suitable for use as a cell conditioning therapy prior to cell transplantation or other suitable therapy. Cell transplantation includes stem cell transplantation, such as hematopoietic stem cell transplantation and bone marrow transplantation. Cell transplantation is allogeneic or autologous. In embodiments, the CAR of the invention eradicates normal cells expressing CD33 or cancer cells expressing CD33, or both, prior to cell transplantation or other suitable therapy. In one aspect, the normal cell expressing CD33 is a myeloid progenitor cell expressing CD33 and the cell transplant is a stem cell transplant.

In one aspect, the invention provides a cell (e.g., an immune effector cell, e.g., a T cell or NK cell, e.g., CART) engineered to express a chimeric antigen receptor of the invention, wherein the cell (e.g., an immune effector cell, e.g., a T cell or NK cell, e.g., "CART") exhibits anti-tumor properties. Accordingly, the invention provides CD 33-CARs comprising a CD33 binding domain and engineered into a cell (e.g., an immune effector cell, e.g., a T cell or NK cell) and methods of their use for adoptive therapy.

In one aspect, the antigen binding domain of the CAR comprises a human CD33 antibody or antibody fragment. In one aspect, the antigen binding domain of the CAR comprises a humanized CD33 antibody or antibody fragment. In one aspect, the antigen binding domain of the CAR comprises a human CD33 antibody fragment comprising an scFv. In one aspect, the antigen binding domain of the CAR is a human CD33 scFv. In one aspect, the antigen binding domain of the CAR comprises a humanized CD33 antibody fragment comprising an scFv. In one aspect, the antigen binding domain of the CAR is a humanized CD33 scFv.

In one aspect, the CD33-CAR comprises at least one intracellular domain, e.g., an intracellular domain described herein, e.g., an intracellular domain selected from the group consisting of a CD137(4-1BB) signaling domain, a CD28 signaling domain, a CD3 zeta signaling domain, and any combination thereof. In one aspect, the CD33-CAR comprises at least one intracellular signaling domain from one or more costimulatory molecules other than CD137(4-1BB) or CD 28.

Chimeric Antigen Receptor (CAR)

The invention also provides a CAR (e.g., a CAR polypeptide) comprising a CD33 binding domain (e.g., a CD33 binding domain as described herein), a transmembrane domain, and an intracellular signaling domain, and wherein the CD33 binding domain comprises heavy chain complementary determining region 1(HC CDR1), heavy chain complementary determining region 2(HC CDR2), and heavy chain complementary determining region 3(HC CDR3) of any heavy chain binding domain amino acid sequence listed in table 2 or table 9. The CD33 binding domain of the CAR may further comprise a light chain complementary determining region 1(LC CDR1), a light chain complementary determining region 2(LC CDR2), and a light chain complementary determining region 3(LC CDR3) of any heavy chain binding domain amino acid sequences listed in table 2 or table 9.

The invention also provides a nucleic acid molecule encoding a CAR as described herein, e.g., encoding a CAR comprising a CD33 binding domain (e.g., as described herein), a transmembrane domain, and an intracellular signaling domain, and wherein the CD33 binding domain comprises heavy chain complementarity determining region 1(HC CDR1), heavy chain complementarity determining region 2(HC CDR2), and heavy chain complementarity determining region 3(HC CDR3) of any heavy chain binding domain amino acid sequence listed in table 2 or table 9. In one embodiment, the encoded CAR CD33 binding domain may further comprise a light chain complementary determining region 1(LC CDR1), a light chain complementary determining region 2(LC CDR2), and a light chain complementary determining region 3(LC CDR3) of any anti-BMCA heavy chain binding domain amino acid sequence listed in table 2 or table 9.

In a particular aspect, the CAR construct of the invention comprises a human scFv domain selected from SEQ ID NOs 39-47, wherein the scFv can be preceded by an optional leader sequence as provided in SEQ ID No. 1 and followed by an optional hinge sequence as provided in SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4 or SEQ ID No. 5, a transmembrane region as provided in SEQ ID No. 6, an intracellular signaling domain comprising SEQ ID No. 7 or SEQ ID No. 8 and a CD3 zeta sequence comprising SEQ ID No. 9 or SEQ ID No. 10, for example, wherein the domains are contiguous with each other and in the same open reading frame to form a single fusion protein. Also included in the invention are nucleotide sequences encoding polypeptides of each of the human scFv fragments selected from the group consisting of SEQ ID NOs 39-47. Also included in the invention are nucleotide sequences encoding polypeptides selected from each of the human scFv fragments of SEQ ID NOs 39-47 and each of the domains of

SEQ ID NOs

1, 2 and 6-9, in addition to the encoded CD33 CAR of the invention.

In one aspect, the exemplary CD33CAR construct comprises an optional leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain. In one aspect, the exemplary CD33CAR construct comprises an optional leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, an intracellular costimulatory domain, and an intracellular stimulatory domain. The specific CD33CAR construct containing the humanized scFv domains of the present invention is provided as SEQ ID NO: 138.

In some embodiments, the full-length CD33CAR sequence is also provided herein as SEQ ID NOs 48-56, as shown in table 2.

An exemplary leader sequence is provided as SEQ ID NO 1. Exemplary hinge/spacer sequences are provided as

SEQ ID NO

2 or 3 or 4 or 5. An exemplary transmembrane domain sequence is provided as SEQ ID NO 6. An exemplary sequence of the intracellular signaling domain of the 4-1BB protein is provided as SEQ ID NO 7. An exemplary sequence of the intracellular signaling domain of CD27 is provided as SEQ ID NO 8. Exemplary CD3 zeta domain sequences are provided as SEQ ID NO 9 or SEQ ID NO 10.

In one aspect, the invention encompasses a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a CD33 binding domain (e.g., as described herein), e.g., contiguous with and in the same open reading frame as a nucleic acid sequence encoding an intracellular signaling domain. In one aspect, the human CD33 binding domain is selected from one or more of SEQ ID NOs 39-47. In one aspect, the human CD33 binding domain is SEQ ID NO 39. In one aspect, the human CD33 binding domain is SEQ ID NO 40. In one aspect, the human CD33 binding domain is SEQ ID NO 41. In one aspect, the human CD33 binding domain is SEQ ID NO 42. In one aspect, the human CD33 binding domain is SEQ ID NO 43. In one aspect, the human CD33 binding domain is SEQ ID NO 44. In one aspect, the human CD33 binding domain is SEQ ID NO 45. In one aspect, the human CD33 binding domain is SEQ ID NO 46. In one aspect, the human CD33 binding domain is SEQ ID NO 47.

In one aspect, the invention encompasses a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a CD33 binding domain, e.g., wherein the sequence is contiguous with and within the same open reading frame as a nucleic acid sequence encoding an intracellular signaling domain. Exemplary intracellular signaling domains that may be used in a CAR include, but are not limited to, one or more intracellular signaling domains such as in CD 3-zeta, CD28, 4-1BB, and the like. In some cases, the CAR can comprise any combination of CD 3-zeta, CD28, 4-1BB, and the like.

In one aspect, the nucleic acid sequence of the CAR construct of the invention is selected from one or more of SEQ ID NOS 75-83. In one aspect, the nucleic acid sequence of the CAR construct comprises (e.g., consists of) SEQ ID NO 75. In one aspect, the nucleic acid sequence of the CAR construct comprises (e.g., consists of) SEQ id no: 76. In one aspect, the nucleic acid sequence of the CAR construct comprises (e.g., consists of) SEQ ID NO 77. In one aspect, the nucleic acid sequence of the CAR construct comprises (e.g., consists of) SEQ ID NO: 78. In one aspect, the nucleic acid sequence of the CAR construct comprises (e.g., consists of) SEQ ID NO: 79. In one aspect, the nucleic acid sequence of the CAR construct comprises (e.g., consists of) SEQ ID NO: 80. In one aspect, the nucleic acid sequence of the CAR construct comprises (e.g., consists of) SEQ ID NO: 81. In one aspect, the nucleic acid sequence of the CAR construct comprises (e.g., consists of) SEQ ID NO: 82. In one aspect, the nucleic acid sequence of the CAR construct comprises (e.g., consists of) SEQ ID NO 83.

Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by deriving the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the nucleic acid of interest may be produced synthetically, rather than cloned. The invention includes retroviral and lentiviral vector constructs that express a CAR that can be directly transduced into a cell.

The invention also includes RNA constructs that can be transfected directly into cells. One method of generating mRNA for transfection involves In Vitro Transcription (IVT) of the template with specially designed primers, followed by addition of poly A, to produce a construct containing 3 ' and 5 ' untranslated sequences ("UTR"), a 5 ' cap and/or an Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a poly adenylated tail (SEQ ID NO:35) of typically 50-2000 bases in length. The RNA thus produced can efficiently transfect cells of various kinds. In one embodiment, the template comprises the sequence of the CAR. In one embodiment, the RNA CAR vector is transduced into a cell by electroporation.

Antigen binding domains

The CAR of the invention comprises a target-specific binding member. The choice of moiety depends on the type and number of ligands that define the surface of the target cell. For example, the antigen binding domain can be selected to recognize an antigen that serves as a cell surface marker on a target cell associated with a particular disease state.

In one aspect, a CAR-mediated T cell response can be directed to an antigen of interest by engineering an antigen binding domain that specifically binds to the desired antigen into the CAR.

In one aspect, the CAR of the invention comprises a binding domain that specifically binds to CD 33. In one aspect, the CAR of the invention comprises an antigen binding domain that specifically binds to human CD 33.

The antigen binding domain may be any protein that binds to an antigen including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including, but not limited to, single domain antibodies, such as the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of camelid-derived nanobodies, as well as alternative scaffolds known in the art to function as antigen binding domains, such as recombinant fibronectin domains and the like. In some cases, it is beneficial for the antigen binding domain to be derived from the same species that will ultimately use the CAR. For example, for use in humans, human or humanized residues comprising the antigen binding domain of an antibody or antibody fragment may be beneficial for the antigen binding domain of a CAR.

In some cases, it is beneficial for the antigen binding domain to be derived from the same species that will ultimately use the CAR. For example, for use in humans, human or humanized residues comprising the antigen binding domain of an antibody or antibody fragment may be beneficial for the antigen binding domain of a CAR. Thus, in one aspect, the antigen binding domain comprises a human antibody or antibody fragment. In one embodiment, a human CD33 binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1(LC CDR1), light chain complementarity determining region 2(LC CDR2), and light chain complementarity determining region 3(LC CDR3) of a human CD33 binding domain described herein, and/or one or more (e.g., all three) heavy chain complementarity determining region 1(HC CDR1), heavy chain complementarity determining region 2(HC CDR2), and heavy chain complementarity determining region 3(HC CDR3) of a human CD33 binding domain described herein, e.g., a human CD33 binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs. In one embodiment, the human CD33 binding domain comprises one or more (e.g., all three) heavy chain complementarity determining region 1(HC CDR1), heavy chain complementarity determining region 2(HC CDR2), and heavy chain complementarity determining region 3(HC CDR3) of a human CD33 binding domain described herein, e.g., human CD33 binding domain has two variable heavy chain regions each comprising HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the human CD33 binding domain comprises a human light chain variable region described herein (e.g., in table 4) and/or a human heavy chain variable region described herein (e.g., in table 3). In one embodiment, the human CD33 binding domain comprises a human heavy chain variable region described herein (e.g., in table 3), such as at least two human heavy chain variable regions described herein (e.g., in table 3). In one embodiment, the CD33 binding domain is an scFv comprising a light chain and a heavy chain of the amino acid sequences of tables 3-4. In one embodiment, a CD33 binding domain (e.g., scFv) comprises a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of the light chain variable region provided in table 4, but no more than 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence having 95-99% identity to the amino acid sequence of table 5; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of a heavy chain variable region provided in table 4, but no more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence having 95-99% identity to an amino acid sequence of table 3. In one embodiment, the human CD33 binding domain comprises a sequence selected from SEQ ID NOS 39-47 or a sequence 95-99% identical thereto. In one embodiment, the human CD33 binding domain is an scFv and the light chain variable region comprising the amino acid sequence described herein (e.g., in table 4) is linked via a linker (e.g., a linker described herein) to the heavy chain variable region comprising the amino acid sequence described herein (e.g., in table 3). In one embodiment, the human CD33 binding domain comprises a (Gly4-Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4(SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can be, for example, in any of the following orientations: a light chain variable region-linker-heavy chain variable region or a heavy chain variable region-linker-light chain variable region.

In one aspect, the antigen binding domain comprises a humanized antibody or antibody fragment. In one embodiment, the humanized CD33 binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1(LC CDR1), light chain complementarity determining region 2(LC CDR2), and light chain complementarity determining region 3(LC CDR3) of a CD33 binding domain described herein, and/or one or more (e.g., all three) heavy chain complementarity determining region 1(HC CDR1), heavy chain complementarity determining region 2(HC CDR2), and heavy chain complementarity determining region 3(HC CDR3) of a CD33 binding domain described herein, e.g., a humanized CD33 binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs. In one embodiment, the humanized CD33 binding domain comprises a (Gly4-Ser) n linker wherein n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4(SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can be, for example, in any of the following orientations: a light chain variable region-linker-heavy chain variable region or a heavy chain variable region-linker-light chain variable region.

In one aspect, a CD33 CAR comprising a humanized CD33 binding domain comprises SEQ ID No. 143. In some aspects, the non-human antibody is humanized, wherein specific sequences or regions of the antibody are modified to increase similarity to an antibody or fragment thereof that occurs naturally in humans. In one aspect, the antigen binding domain is humanized. Examples of humanized CD33 antibodies for use with the CART described herein include hp67.6 or Gemtuzumab (Gemtuzumab), as described in WO 2012123755.

Humanized antibodies can be generated using a variety of techniques known in the art, including, but not limited to, CDR-grafting (see, e.g., European patent No. EP 239,400; International publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101 and 5,585,089, each of which is incorporated herein by reference in its entirety), veneering or resurfacing (see, e.g., European patent No. EP 592,106 and EP 519,596; Padlan,1991, Molecular Immunology,28(4/5): 489-498; studnika et al, 1994, Protein Engineering,7(6): blood 814; and Roskoa et al, 1994, PNAS,91:969-973, each of which is incorporated herein by reference in its entirety), chain shuffling (see, e.g., U.S. Pat. No. 5,565,805, which is incorporated herein by reference in its entirety) and techniques disclosed in, e.g., in the following: U.S. patent application publication No. US 2005/0042664, U.S. patent application publication No. US 2005/0048617, U.S. patent No. 6,407,213, U.S. patent No. 5,766,886, international publication No. WO 9317105, Tan et al, j.immunol.,169:1119-25(2002), Caldas et al, Protein eng, 13(5):353-60(2000), Morea et al, Methods,20(3):267-79(2000), Baca et al, j.biol.chem.,272(16):10678-84(1997), Roguska et al, Protein Eng.,9(10):895-904(1996), Couto et al, Cancer Res.,55(23Supp):5973s-5977s (1995), Couto et al, Cancer Res.,55(8):1717-22(1995), Sandhu J S, Gene,150(2):409-10(1994) and Pedersen et al, j.mol.biol.,235(3):959-73(1994), each of which is incorporated by reference herein in its entirety. Often, framework residues in the framework regions will be replaced with corresponding residues from the CDR donor antibody to alter, e.g., improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction of the CDRs and framework residues to identify framework residues important for antigen binding and performing sequence comparisons to identify unusual framework residues at specific positions. (see, e.g., Queen et al, U.S. Pat. No. 5,585,089; and Riechmann et al, 1988, Nature,332:323, which are incorporated herein by reference in their entirety).

The humanized antibody or antibody fragment has one or more amino acid residues from a non-human source left therein. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. As provided herein, a humanized antibody or antibody fragment comprises one or more CDRs from a non-human immunoglobulin molecule and a framework region, wherein the amino acid residues comprising the framework are fully or mostly derived from a human germline. A number of techniques for humanizing antibodies or antibody fragments are well known in the art and can be performed by replacing the corresponding human antibody sequence with multiple CDR sequences or one CDR sequence of rodents, essentially as described by Winter and co-workers (Jones et al, Nature,321:522-525 (1986); Reichmann et al, Nature,332:323-327 (1988); Verhoeyen et al, Science,239:1534-1536(1988)), i.e., CDR grafting methods (EP 239,400; PCT publication WO 91/09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference in their entirety). In such humanized antibodies and antibody fragments, regions substantially smaller than an intact human variable domain have been replaced by corresponding sequences from a non-human species. Humanized antibodies are often human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments may also be achieved by veneering or resurfacing (EP 592,106; EP519,596; Padlan,1991, Molecular Immunology,28(4/5): 489-498; Studnica et al, Protein Engineering,7(6):805-814 (1994); and Roguska et al, PNAS,91:969-973(1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

The human variable domains (light and heavy chain variable domains) to be used to generate the humanized antibody are selected for reducing antigenicity. The sequence of the variable domains of rodent antibodies is screened against a complete library of known human variable domain sequences according to the so-called "best fit" method. The human sequence closest to the rodent sequence was then identified and accepted as the human Framework Region (FR) for the humanized antibody (Sims et al, J.Immunol,151:2296 (1993); Chothia et al, J.mol.biol.,196:901(1987), the contents of which are incorporated herein by reference in their entirety). Another approach uses a specific framework derived from the consensus sequence of all human antibodies with a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (see, e.g., Nicholson et al, mol. Immun.34(16-17):1157-1165 (1997); Carter et al, Proc. Natl. Acad. Sci. USA,89:4285 (1992); Presta et al, J.Immunol.,151:2623(1993), the contents of which are incorporated herein by reference in their entirety). In some embodiments, the framework regions (e.g., all four framework regions) of the heavy chain variable region are derived from the VH4_4-59 germline sequence. In one embodiment, the framework region may comprise one, two, three, four or five modifications, e.g., substitutions, e.g., amino acids from the corresponding murine sequence (e.g., SEQ ID NO: 138). In one embodiment, the framework regions (e.g., all four framework regions) of the light chain variable region are derived from the VK3 — 1.25 germline sequence. In one embodiment, the framework region may comprise one, two, three, four or five modifications, e.g., substitutions, e.g., amino acids from the corresponding murine sequence (e.g., SEQ ID NO: 138).

In some aspects, portions of the CAR compositions of the invention comprising antibody fragments are humanized, retaining high affinity for the target antigen and other favorable biological properties. According to one aspect of the invention, humanized antibodies and antibody fragments are prepared by a method of analyzing the parent sequence and various conceptual humanized products using three-dimensional models of the parent sequence and the humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these display results allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., analysis of residues that affect the ability of the candidate immunoglobulin to bind the target antigen. In this manner, FR residues can be selected and combined from the acceptor and import sequences to achieve a desired antibody or antibody fragment characteristic, such as increased affinity for the target antigen. Generally, CDR residues are directly and most substantially involved in affecting antigen binding.

The humanized antibody or antibody fragment may retain antigen specificity similar to the original antibody (e.g., in the present invention), the ability to bind human CD33 or a fragment thereof. In some embodiments, the humanized antibody or antibody fragment may have improved affinity and/or specificity for binding to human CD33 or a fragment thereof.

In one aspect, the antigen binding domain portion comprises one or more sequences selected from SEQ ID NOS 39-47. In one aspect, a CD33 CAR comprising a human CD33 binding domain is selected from one or more sequences selected from SEQ ID NOs 48-56.

In one aspect, the CD33 binding domain is characterized by a particular functional characteristic or property of an antibody or antibody fragment. For example, in one aspect, the antigen binding domain-containing portion of the CAR composition of the invention specifically binds human CD33 or a fragment thereof. In one aspect, the invention relates to an antigen binding domain comprising an antibody or antibody fragment, wherein the antibody binding domain specifically binds to CD33 protein or a fragment thereof, wherein the antibody or antibody fragment comprises a variable light chain and/or a variable heavy chain comprising the amino acid sequence of SEQ ID NOS 48-56. In one aspect, the antigen binding domain comprises the amino acid sequence of an scFv selected from the group consisting of SEQ ID NOS 39-47. In certain aspects, the scFv is contiguous with and within the same open reading frame as the leader sequence. In one aspect, the leader sequence is the polypeptide sequence provided as SEQ ID NO 1.

In one aspect, the CD33 binding domain is a fragment, e.g., a single chain variable fragment (scFv). In one aspect, the CD33 binding domain is Fv, Fab, (Fab') ₂Or bifunctional (e.g., bispecific) hybrid antibodies (e.g., Lanzavecchia et al, Eur. J. Immunol.17,105 (1987)). In one aspect, the antibodies of the invention andfragments thereof bind to the CD33 protein or fragment thereof with wild-type or enhanced affinity.

In some cases, human scFv can be derived from a display library. A display library is a set of entities; each entity includes an accessible polypeptide component and a recoverable component that encodes or identifies the polypeptide component. The polypeptide components are so altered as to represent different amino acid sequences. The polypeptide component can be any length, for example from three amino acids to over 300 amino acids. The display library entity may comprise more than one polypeptide component, e.g. two polypeptide chains of a Fab. In an exemplary embodiment, a display library may be used to identify human CD33 binding domains. In selection, the polypeptide component of each library member is probed with CD33 or a fragment thereof, and if the polypeptide component binds to CD33, the display library member is identified, typically by resting on a support.

The remaining display library members are recovered from the support and analyzed. Analysis may include amplification and subsequent selection under similar or dissimilar conditions. For example, positive and negative selections may be alternated. The analysis may also include determining the amino acid sequence of the polypeptide component (i.e., the CD33 binding domain) and purifying the polypeptide component for detailed characterization.

A variety of formats can be used to display the library. Examples include phage display. In phage display, the protein component is typically covalently linked to a phage coat protein. The linkage is generated by translation of a nucleic acid encoding a protein component fused to the coat protein. The linkage may include a flexible peptide linker, a protease site, or an amino acid that is incorporated due to the suppression of a stop codon. Phage display is described, for example, in U.S. Pat. nos. 5,223,409; smith (1985) Science 228: 1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; de Haard et al (1999) J.biol.chem 274: 18218-30; hoogenboom et al (1998) Immunotechnology 4: 1-20; hoogenboom et al (2000) Immunol Today 2:371-8 and Hoet et al (2005) Nat Biotechnol.23(3) 344-8. Phage displaying the protein component can be grown and harvested from the growth medium using standard preparation methods for phage (e.g., PEG precipitation). After selection of each display phage, the nucleic acid encoding the selected protein component can be isolated after amplification from the selected phage-infected cell or from the phage itself. Single colonies or plaques may be picked, nucleic acids isolated and sequenced.

Other display modes include cell-based display (see, e.g., WO 03/029456), protein-nucleic acid fusion (see, e.g., U.S. Pat. No. 6,207,446), ribosome display (see, e.g., Mattheakis et al, (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes et al, (2000) Nat Biotechnol.18: 1287-92; Hanes et al, (2000) Methods enzymol.328: 404-30; and Schaffitzel et al, (1999) J Immunol methods.231(1-2):119-35) and E.coli periplasmic display 2005 ((Nov 22; PMID: 16337958).

In some cases, scFv can be prepared according to methods known in the art (see, e.g., Bird et al, (1988) Science 242: 423-. The scFv molecule can be produced by linking the VH and VL regions together using a flexible polypeptide linker. ScFv molecules comprise linkers optimized for length and/or amino acid composition (e.g., Ser-Gly linkers). Linker length can greatly influence how the variable regions of the scFv fold and interact. Indeed, if short polypeptide linkers (e.g., between 5-10 amino acids) are used, intra-chain folding is prevented. Interchain folding is also required so that the two variable regions together form a functional epitope binding site. For examples of linker orientation and size, see, e.g., Hollinger et al, 1993Proc Natl Acad.Sci.U.S.A.90: 6444-.

The scFv may comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises the amino acids glycine and serine. In another embodimentIn (2), the linker sequence comprises a set of glycine and serine repeats such as (Gly)₄Ser)_nWherein n is a positive integer equal to or greater than 1 (SEQ ID NO: 25). In one embodiment, the linker may be (Gly4Ser)₄(SEQ ID NO:27) or (Gly4Ser)₃(SEQ ID NO: 28). Variability in linker length can preserve or enhance activity, resulting in superior efficacy in activity studies.

Exemplary human CD33 CAR constructs and antigen binding domains

Exemplary CD33 CAR constructs disclosed herein comprise scFv (e.g., human scFv as disclosed in tables 2 and 9 herein, optionally preceded by an optional leader sequence (e.g., exemplary leader amino acid sequence and nucleotide sequence are SEQ ID NO:1 and SEQ ID NO:12, respectively.) the sequences of the human scFv fragments are provided in Table 2 herein (SEQ ID NO:39-83, comprising an optional leader sequence). the sequences of the human scFv fragments without leader sequence are provided in Table 9 herein (nucleotide sequence is SEQ ID NO:255-261, and amino acid sequence is SEQ ID NO: 262-268). the CD33 CAR construct may further comprise an optional hinge domain, e.g., a CD8 hinge domain (e.g., comprising the amino acid sequence of SEQ ID NO:2 or encoded by the nucleic acid sequence of SEQ ID NO: 1), for example, a CD8 transmembrane domain (e.g., comprising the amino acid sequence of SEQ ID NO:6 or encoded by the nucleotide sequence of SEQ ID NO: 17); an intracellular domain, e.g., a 4-1BB intracellular domain (e.g., comprising the amino acid sequence of SEQ ID NO:7 or encoded by the nucleotide sequence of SEQ ID NO: 18); and a functional signaling domain, e.g., a CD3 zeta domain (e.g., comprising the amino acid sequence of SEQ ID NO:9 or 10, or encoded by the nucleotide sequence of SEQ ID NO:20 or 21). In certain embodiments, the domains are contiguous and in the same open reading frame to form a single fusion protein. In other embodiments, each domain is in an isolated polypeptide, e.g., as in an RCAR molecule as described herein.

In certain embodiments, the full length CD33 CAR molecule comprises the amino acid sequence of or is encoded by the amino acid sequence of CAR33-1, CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9 provided in table 2, or a sequence that is substantially identical (e.g., 95-99%) thereto, or the nucleotide sequence of or is substantially identical (e.g., 95-99%) thereto of CAR33-1, CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9 provided in table 2.

In certain embodiments, the CD33 CAR molecule or anti-CD 33 antigen binding domain comprises the scFv amino acid sequence (with or without a leader sequence) of CAR33-1, CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9 provided in table 2; or a sequence comprising or encoded by an scFv amino acid sequence of CAR33-1, CAR33-2, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-9 provided in table 9, or encoded by a nucleotide sequence of CAR33-1, CAR33-2, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-9 provided in table 9, or a sequence that is substantially identical (e.g., 95-99% identical, or up to 20, 15, 10, 8, 6, 5, 4, 3, 2, or 1 amino acid change, e.g., a substitution (e.g., a conservative substitution)).

In certain embodiments, a CD33 CAR molecule or anti-CD 33 antigen binding domain comprises a CAR33-1, CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, the heavy chain variable region and/or the light chain variable region of CAR33-9, provided in table 2, or a sequence that is substantially identical (e.g., 95-99% identical, or up to 20, 15, 10, 8, 6, 5, 4, 3, 2, or 1 amino acid change, e.g., a substitution (e.g., a conservative substitution)) to any of the foregoing sequences.

In certain embodiments, the CD33 CAR molecule or anti-CD 33 antigen binding domain comprises 1, 2, or 3 CDRs from a heavy chain variable region (e.g., HC CDR1, HC CDR2, and/or HC CDR3) provided in table 3; and/or 1, 2, or 3 CDRs from a light chain variable region of CAR33-1, CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9 (e.g., LC CDR1, LC CDR2, and/or LC CDR3) provided in table 4; or a sequence that is substantially identical (e.g., 95-99% identical, or up to 5, 4, 3, 2, or 1 amino acid change, e.g., a substitution (e.g., a conservative substitution)) to any of the sequences described above.

In certain embodiments, the CD33 CAR molecule or anti-CD 33 antigen binding domain comprises 1, 2, or 3 CDRs from a heavy chain variable region (e.g., HC CDR1, HC CDR2, and/or HC CDR3) provided in table 10; and/or 1, 2, or 3 CDRs from a light chain variable region of CAR33-1, CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9 (e.g., LC CDR1, LC CDR2, and/or LC CDR3) provided in table 11; or a sequence that is substantially identical (e.g., 95-99% identical, or up to 5, 4, 3, 2, or 1 amino acid change, e.g., a substitution (e.g., a conservative substitution)) to any of the sequences described above.

In certain embodiments, the CD33 CAR molecule or anti-CD 33 antigen binding domain comprises 1, 2, or 3 CDRs from a heavy chain variable region (e.g., HC CDR1, HC CDR2, and/or HC CDR3) provided in table 12; and/or 1, 2, or 3 light chain variable regions from CAR33-1, CAR33-2, CAR33-3, CAR33-4, CAR33-5, CAR33-6, CAR33-7, CAR33-8, CAR33-9 (e.g., LCDR1, LCDR2, and/or LCDR3) provided in table 13; or a sequence that is substantially identical (e.g., 95-99% identical, or up to 5, 4, 3, 2, or 1 amino acid change, e.g., a substitution (e.g., a conservative substitution)) to any of the sequences described above.

The human CDR sequences of the scFv domains are shown in table 3 for the heavy chain variable domains, and in table 4 for the light chain variable domains. "ID" represents the corresponding SEQ ID NO for each CDR. The CDRs provided in tables 3 and 4 are numbered jointly according to the Kabat and Chothia numbering schemes.

TABLE 3 heavy chain variable Domain CDR

Candidates

HCDR1

ID

HCDR2

ID

HCDR3

ID

CAR33-1

GYSFTSYWIG

84

IIYPGDSDTRYSPSFQG

93

LGGSLPDYGMDV

102

CAR33-2

GYIFTNYYVH

85

IISPSGGSPTYAQRLQG

94

ESRLRGNRLGLQSSIFDH

103

CAR33-3

GFTFSSYAMS

86

AISGSGGSTYYADSVKG

95

EDTIRGPNYYYYGMDV

104

CAR33-4

GYSFTSYWIG

87

IIYPGDSDTRYSPSFQG

96

GGYSDYDYYFDF

105

CAR33-5

GFTFDDYAMH

88

VIWPDGGQKYYGDSVKG

97

HFNAWDY

106

CAR33-6

GFTFSIFAMH

89

TISYDGSNAFYADSVEG

98

AGDGGYDVFDS

107

CAR33-7

GFTFSSYAMS

90

AISGSGGSTYYADSVKG

99

ETDYYGSGTFDY

108

CAR33-8

GYMFTDFFIH

91

WINPNSGVTKYAQKFQG

100

WYSSGWYGIANI

109

CAR33-9

GYSFTNYWIG

92

IIYPGDSDTRYSPSFQG

101

HGPSSWGEFDY

110

TABLE 4 light chain variable Domain CDR

Candidates

LCDR1

ID

LCDR2

ID

LCDR3

ID

CAR33-1

RSSQSLLHSNGYNYLD

111

LGSNRAS

120

MQALQTLIT

129

CAR33-2

QASQDINNHLN

112

DTSNLEI

121

QQYENLPLT

130

CAR33-3

RASQDIDTWLA

113

AASNLQG

122

QQASIFPPT

131

CAR33-4

RSSQSLLHSNGYNYLD

114

LGSNRAS

123

MQALQTPFT

132

CAR33-5

QASQGISQFLN

115

DASNLEP

124

QQYDDLPLT

133

CAR33-6

RSSQSLLHSNGYNYLD

116

LGSNRAS

125

MQALQTPT

134

CAR33-7

RASQGIGIYLA

117

GASTLQS

126

QQSNNFPPT

135

CAR33-8

QASHDISNYLH

118

DASNLET

127

QQSDDLPHT

136

CAR33-9

RASQSISSYLN

119

AASSLQS

128

QQSYSTPLT

137

Table 10: heavy chain variable Domain CDRs according to the Kabat numbering scheme (Kabat et al (1991), Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md.)

Candidates

HCDR1

ID

HCDR2

ID

HCDR3

ID

141643-CAR33-1

SYWIG

269

IIYPGDSDTRYSPSFQG

278

LGGSLPDYGMDV

287

141644-CAR33-2

NYYVH

270

IISPSGGSPTYAQRLQG

279

ESRLRGNRLGLQSSIFDH

288

141645-CAR33-3

SYAMS

271

AISGSGGSTYYADSVKG

280

EDTIRGPNYYYYGMDV

289

141646-CAR33-4

SYWIG

272

IIYPGDSDTRYSPSFQG

281

GGYSDYDYYFDF

290

141647-CAR33-5

DYAMH

273

VIWPDGGQKYYGDSVKG

282

HFNAWDY

291

141648-CAR33-6

IFAMH

274

TISYDGSNAFYADSVEG

283

AGDGGYDVFDS

292

141649-CAR33-7

SYAMS

275

AISGSGGSTYYADSVKG

284

ETDYYGSGTFDY

293

141650-CAR33-8

DFFIH

276

WINPNSGVTKYAQKFQG

285

WYSSGWYGIANI

294

141651-CAR33-9

NYWIG

277

IIYPGDSDTRYSPSFQG

286

HGPSSWGEFDY

295

Table 11: light chain variable Domain CDRs according to the Kabat numbering scheme (Kabat et al (1991), Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md.)

Candidates

LCDR1

ID

LCDR2

ID

LCDR3

ID

141643-CAR33-1

RSSQSLLHSNGYNYLD

296

LGSNRAS

305

MQALQTLIT

314

141644-CAR33-2

QASQDINNHLN

297

DTSNLEI

306

QQYENLPLT

315

141645-CAR33-3

RASQDIDTWLA

298

AASNLQG

307

QQASIFPPT

316

141646-CAR33-4

RSSQSLLHSNGYNYLD

299

LGSNRAS

308

MQALQTPFT

317

141647-CAR33-5

QASQGISQFLN

300

DASNLEP

309

QQYDDLPLT

318

141648-CAR33-6

RSSQSLLHSNGYNYLD

301

LGSNRAS

310

MQALQTPT

319

141649-CAR33-7

RASQGIGIYLA

302

GASTLQS

311

QQSNNFPPT

320

141650-CAR33-8

QASHDISNYLH

303

DASNLET

312

QQSDDLPHT

321

141651-CAR33-9

RASQSISSYLN

304

AASSLQS

313

QQSYSTPLT

322

Table 12: heavy chain variable domain CDRs according to the Chothia numbering scheme (Al-Lazikani et Al, (1997) JMB 273,927-948)

Candidates

HCDR1

ID

HCDR2

ID

HCDR3

ID

141643-CAR33-1

GYSFTSY

323

YPGDSD

332

LGGSLPDYGMDV

341

141644-CAR33-2

GYIFTNY

324

SPSGGS

333

ESRLRGNRLGLQSSIFDH

342

141645-CAR33-3

GFTFSSY

325

SGSGGS

334

EDTIRGPNYYYYGMDV

343

141646-CAR33-4

GYSFTSY

326

YPGDSD

335

GGYSDYDYYFDF

344

141647-CAR33-5

GFTFDDY

327

WPDGGQ

336

HFNAWDY

345

141648-CAR33-6

GFTFSIF

328

SYDGSN

337

AGDGGYDVFDS

346

141649-CAR33-7

GFTFSSY

329

SGSGGS

338

ETDYYGSGTFDY

347

141650-CAR33-8

GYMFTDF

330

NPNSGV

339

WYSSGWYGIANI

348

141651-CAR33-9

GYSFTNY

331

YPGDSD

340

HGPSSWGEFDY

349

Table 13: light chain variable domain CDRs according to the Chothia numbering scheme (Al-Lazikani et Al, (1997) JMB 273,927-948)

Candidates

LCDR1

ID

LCDR2

ID

LCDR3

ID

141643-CAR33-1

SQSLLHSNGYNY

350

LGS

359

ALQTLI

368

141644-CAR33-2

SQDINNH

351

DTS

360

YENLPL

369

141645-CAR33-3

SQDIDTW

352

AAS

361

ASIFPP

370

141646-CAR33-4

SQSLLHSNGYNY

353

LGS

362

ALQTPF

371

141647-CAR33-5

SQGISQF

354

DAS

363

YDDLPL

372

141648-CAR33-6

SQSLLHSNGYNY

355

LGS

364

ALQTP

373

141649-CAR33-7

SQGIGIY

356

GAS

365

SNNFPP

374

141650-CAR33-8

SHDISNY

357

DAS

366

SDDLPH

375

141651-CAR33-9

SQSISSY

358

AAS

367

SYSTPL

376

In certain embodiments, a CAR molecule (e.g., a CAR nucleic acid or a CAR polypeptide) or CD33 binding domain described herein comprises:

(1) one, two or three Light Chain (LC) CDRs selected from one of:

(2) One, two or three Heavy Chain (HC) CDRs from one of:

(i) HC CDR1 of SEQ ID NO:84, HC CDR2 of SEQ ID NO:93 and HC CDR3 of SEQ ID NO:102 of CAR 33-1;

(ii) HC CDR1 of SEQ ID NO 85, HC CDR2 of SEQ ID NO 94 and HC CDR3 of SEQ ID NO 103 of CAR 33-2;

(iii) HC CDR1 of SEQ ID NO 86, HC CDR2 of SEQ ID NO 95, and HC CDR3 of SEQ ID NO 104 of CAR 33-3;

(iv) HC CDR1 of SEQ ID NO 87, HC CDR2 of SEQ ID NO 96 and HC CDR3 of SEQ ID NO 105 of CAR 33-4;

(iv) HC CDR1 of SEQ ID NO:88, HC CDR2 of SEQ ID NO:97 and HC CDR3 of SEQ ID NO:106 of CAR 33-5;

(vi) HC CDR1 of SEQ ID NO. 89, HC CDR2 of SEQ ID NO. 98 and HC CDR3 of SEQ ID NO. 107 of CAR 33-6;

(vii) HC CDR1 of SEQ ID NO:90, HC CDR2 of SEQ ID NO:99, and HC CDR3 of SEQ ID NO:108 of CAR 33-7;

(viii) HC CDR1 of SEQ ID NO 91, HC CDR2 of SEQ ID NO 100 and HC CDR3 of SEQ ID NO 109 of CAR 33-8; or

(ix) HC CDR1 of SEQ ID NO 92, HC CDR2 of SEQ ID NO 101, and HC CDR3 of SEQ ID NO 110 of CAR 33-9.

In certain embodiments, a CAR molecule (e.g., a CAR nucleic acid or a CAR polypeptide) described herein comprises:

(1) one, two or three Light Chain (LC) CDRs selected from one of:

(ii) LC CDR1 of SEQ ID NO:297, LC CDR2 of SEQ ID NO:306, and LC CDR3 of SEQ ID NO:315 of CAR 33-2;

(2) One, two or three Heavy Chain (HC) CDRs selected from one of:

(i) HC CDR1 of SEQ ID NO:269, HC CDR2 of SEQ ID NO:278, and HC CDR3 of SEQ ID NO:287 of CAR 33-1;

(ii) HC CDR1 of SEQ ID NO:270, HC CDR2 of SEQ ID NO:279, and HC CDR3 of SEQ ID NO:288 of CAR 33-2;

(iii) HC CDR1 of SEQ ID NO:271, HC CDR2 of SEQ ID NO:280 and HC CDR3 of SEQ ID NO:289 of CAR 33-3;

(iv) HC CDR1 of SEQ ID NO:272, HC CDR2 of SEQ ID NO:281, and HC CDR3 of SEQ ID NO:290 of CAR 33-4;

(iv) HC CDR1 of SEQ ID NO:273, HC CDR2 of SEQ ID NO:282, and HC CDR3 of SEQ ID NO:291 of CAR 33-5;

(vi) HC CDR1 of SEQ ID NO:274, HC CDR2 of SEQ ID NO:283, and HC CDR3 of SEQ ID NO:292 of CAR 33-6;

(vii) HC CDR1 of SEQ ID NO:275, HC CDR2 of SEQ ID NO:284, and HC CDR3 of SEQ ID NO:293 of CAR 33-7;

(viii) HC CDR1 of SEQ ID NO:276, HC CDR2 of SEQ ID NO:285 and HC CDR3 of SEQ ID NO:294 of CAR 33-8; or

(ix) HC CDR1 of SEQ ID NO 277, HC CDR2 of SEQ ID NO 286, and HC CDR3 of SEQ ID NO 295 of CAR 33-9.

(1) One, two or three Light Chain (LC) CDRs selected from one of:

(2) One, two or three Heavy Chain (HC) CDRs selected from one of:

In embodiments, a fully human anti-CD 33 single chain variable fragment (scFv) was generated and cloned into a lentiviral expression vector along with the intracellular CD3 zeta chain and the intracellular costimulatory domain of 4-1 BB. The names of exemplary fully human anti-CD 33scFv are described in table 1.

In embodiments, the order in which the VL domain and VH domain occur in the scFv varies (i.e., VL-VH, or VH-VL orientation), and wherein 3 or 4 copies of a "G4S" (SEQ ID NO:25) subunit (wherein each subunit comprises the sequence GGGGS (SEQ ID NO:25) (e.g., (G4S) ₃(SEQ ID NO:28) or (G4S)₄(SEQ ID NO:27)) to generate the integrity of the scFv domains as shown in Table 3.

Exemplary sequences of human scFv fragments (SEQ ID NOS: 39-83, including optional leader sequences) are provided in Table 2 herein. It should be noted that the present invention also encompasses scFv fragments of SEQ ID NOs 39-83 without leader sequences (e.g., without the amino acid sequence of SEQ ID NO:1 or the nucleotide sequence of SEQ ID NO: 12). Exemplary sequences of human scFv fragments without leader sequences are provided in Table 9 herein (nucleotide sequence SEQ ID NO:255-261, and amino acid sequence SEQ ID NO: 262-268).

Leader sequence (amino acid sequence) (SEQ ID NO:1)

MALPVTALLLPLALLLHAARP

Leader sequence (nucleic acid sequence) (SEQ ID NO:12)

ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCC

CD8 hinge (amino acid sequence) (SEQ ID NO:2)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

CD8 hinge (nucleic acid sequence) (SEQ ID NO:13)

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

CD8 transmembrane region (amino acid sequence) (SEQ ID NO:6)

IYIWAPLAGTCGVLLLSLVITLYC

CD8 transmembrane (nucleic acid sequence) (SEQ ID NO:17)

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

4-1BB intracellular domain (amino acid sequence) (SEQ ID NO:7)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

4-1BB intracellular domain (nucleic acid sequence) (SEQ ID NO:18)

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

CD28 intracellular domain (amino acid sequence) (SEQ ID NO:379)

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:379)

CD28 intracellular domain (nucleotide sequence) (SEQ ID NO:380)

AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ IDNO:380)

ICOS intracellular Domain (amino acid sequence) (SEQ ID NO:381)

T K K K Y S S S V H D P N G E Y M F M R A V N T A K K S R L T D V T L(SEQID NO:381)

ICOS intracellular domain (nucleotide sequence) (SEQ ID NO:382)

ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTA(SEQ ID NO:382)

CD3 zeta Domain (amino acid sequence) (SEQ ID NO:9)

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD3 zeta (nucleic acid sequence) (SEQ ID NO:20)

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

CD3 zeta Domain (amino acid sequence; NCBI reference sequence NM-000734.3) (SEQ ID NO:10)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD3 ζ (nucleic acid sequence; NCBI reference sequence NM-000734.3); (SEQ ID NO:21)

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAG

AACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT

TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGA

AGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGG

AGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC

ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC

CCTTCACATGCAGGCCCTGCCCCCTCGC

IgG4 hinge (amino acid sequence) (SEQ ID NO:36)

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM

IgG4 hinge (nucleotide sequence) (SEQ ID NO:37)

GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG

In embodiments, these clones contain a Q/K residue change in the signal domain derived from the costimulatory domain of the CD3 zeta chain.

Table 2: human CD33 CAR constructs

TABLE 9 human CD33 CAR scFv sequences

In embodiments, the CAR scFv fragment is cloned into a lentiviral vector to generate a full length CAR construct in a single coding open reading frame and using a promoter for expression (e.g., the EF1 a promoter) (SEQ ID NO: 11).

11EF1 alpha promoter of SEQ ID NO

CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA

Gly/Ser(SEQ ID NO:25)

GGGGS

Gly/Ser(SEQ ID NO:26)：

This sequence may contain 1-6 "Gly Gly Gly Gly Ser" repeat units

GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS

Gly/Ser(SEQ ID NO:27)

GGGGSGGGGS GGGGSGGGGS

Gly/Ser(SEQ ID NO:28)

GGGGSGGGGS GGGGS

Gly/Ser(SEQ ID NO:29)

GGGS

PolyA：(A)₅₀₀₀(SEQ ID NO:30)

This sequence may contain 50-5000 adenines.

PolyA：(T)₁₀₀(SEQ ID NO:31)

PolyA：(T)₅₀₀₀(SEQ ID NO:32)

This sequence may comprise 50-5000 thymines.

PolyA：(A)₅₀₀₀(SEQ ID NO:33)

This sequence may comprise 100-5000 adenines.

PolyA：(A)₄₀₀(SEQ ID NO:34)

PolyA：(A)₂₀₀₀(SEQ ID NO:35)

Gly/Ser (SEQ ID NO: 38). The sequence may comprise 1-10 "Gly Gly Gly Ser" repeat units

GGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS

Additional examples of CAR molecules or antibody fragments thereof are provided in example 3. anti-CD 33 antibody 2213 is disclosed in murine and humanized forms. For example, example 3 provides the following: 2213 nucleotide sequence of murine anti-CD 33IgG4 (SEQ ID NO: 138); 2213 CAR nucleotide sequence (SEQ ID NO: 139); 2213 CAR amino acid sequence (SEQ ID NO: 140); 2213scFv nucleotide sequence (SEQ ID NO: 141); and 2213scFv amino acid sequence (SEQ ID NO: 142); 2218 humanized anti-CD 33IgG4H nucleotide sequence (SEQ ID NO: 143).

Other embodiments disclosed in example 3 include the CAR molecule of Gemtuzumab ozogamicin (Gemtuzumab ozogamicin) previously sold as milotat (Mylotarg) and anti-CD 33 antibody fragments (e.g., the humanized form described herein as "humanized my 96"). The amino acid sequence of the anti-CD 33scFv of gemtuzumab ozogamicin (an immunoconjugate targeting CD 33) with 41BB and CD3 zeta signaling domains is described in example 3 and SEQ ID NO: 145. The corresponding nucleotide sequence of humanized my96 is depicted as SEQ ID NO: 144. The humanized my96 nucleotide sequence is provided herein as SEQ ID NO:146 and the amino acid sequence is SEQ ID NO: 147.

In one embodiment, the CD33 CARs and CD33 CART described herein comprise an antigen binding domain comprising one or more (e.g., one, two, or three) CDRs of a heavy chain variable domain and/or one or more (e.g., one, two, or three) CDRs of a light chain variable domain, or VH or VL of a scFv sequence encoded by GenBank reference AM402974.1 (see Wang et al mol. ther., vol. 23: 1, page 184-191 (2015), which is hereby incorporated by reference).

The CAR scFv fragment can be cloned into a lentiviral vector to generate a full length CAR construct in a single coding open reading frame and using the EF1 a promoter for expression (SEQ ID NO: 11).

The CAR construct may include a Gly/Ser linker having one or more of the following sequences: GGGGS (SEQ ID NO: 25); comprises 1-6 "Gly Gly Gly Gly Ser" repeat units, e.g., GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS (SEQ ID NO: 26); GGGGSGGGGS GGGGSGGGGS (SEQ ID NO: 27); GGGGSGGGGS GGGGS (SEQ ID NO: 28); GGGS (SEQ ID NO: 29); or comprises 1-10 "Gly Gly Gly Ser" repeat units, e.g., GGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS (SEQ ID NO: 38). In embodiments, the CAR construct comprises a polyadenylation sequence, e.g., a sequence comprising 50-5000 or 100-5000 adenine (e.g., SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:35), or a sequence comprising 50-5000 thymines (e.g., SEQ ID NO:31, SEQ ID NO: 32). Alternatively, the CAR construct may, for example, comprise a linker comprising sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO:383)

Bispecific CAR

In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies are specific for no more than two antigens. Bispecific antibody molecules are characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen (e.g., the same protein (or subunits of a multimeric protein)). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes are non-overlapping. In one embodiment, the first and second epitopes are on different antigens (e.g., different proteins (or different subunits of a multimeric protein)). In one embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In one embodiment the bispecific antibody molecule comprises a half-antibody having binding specificity for a first epitope and a half-antibody having binding specificity for a second epitope. In one embodiment the bispecific antibody molecule comprises a half-antibody or fragment thereof having binding specificity for a first epitope and a half-antibody or fragment thereof having binding specificity for a second epitope. In one embodiment the bispecific antibody molecule comprises a scFv or fragment thereof having binding specificity for a first epitope and a scFv or fragment thereof having binding specificity for a second epitope.

In certain embodiments, the antibody molecule is a multispecific (e.g., bispecific or trispecific) antibody molecule. Protocols for producing bispecific or heterodimeric antibody molecules are known in the art; including, but not limited to, for example, a "knot in knot" solution, such as described in US 5731168; electrostatically-directed Fc pairing, for example, as described in WO 09/089004, WO 06/106905, and WO 2010/129304; strand Exchange Engineered Domain (SEED) heterodimer formation, e.g., as described in WO 07/110205; fab arm exchange, e.g., as described in WO 08/119353, WO 2011/131746 and WO 2013/060867; diabody conjugates are crosslinked by antibodies to produce bispecific structures, e.g., using heterobifunctional reagents having amine-reactive groups and sulfhydryl-reactive groups, e.g., as described in US 4433059; bispecific antibody determinants produced by: recombination of half-antibodies (heavy chain-light chain pairs or fabs) from different antibodies by means of cycles of reduction and oxidation of the disulfide bond between the two heavy chains, e.g. as described in US 4444878; trifunctional antibodies, e.g., three Fab fragments cross-linked by thiol-reactive groups, e.g., as described in US 5273743; a biosynthetic binding protein, e.g., a pair of scFvs cross-linked by a C-terminal tail, preferably by disulfide bonds or amine reactive chemical cross-linking, e.g., as described in US 5534254; bifunctional antibodies, e.g., Fab fragments with different binding specificities dimerized by leucine zippers (e.g., c-fos and c-jun) that have replaced constant domains, e.g., as described in US 5582996; bispecific and oligospecific monovalent and oligovalent receptors, e.g., the VH-CH1 regions of two antibodies (two Fab fragments) linked via a polypeptide spacer sequence between the CH1 region of one antibody and the VH region of the other antibody (typically with an associated light chain), e.g., as described in US 5591828; bispecific DNA-antibody conjugates, e.g., antibodies or Fab fragments cross-linked by a double-stranded DNA fragment, e.g., as described in US 5635602; bispecific fusion proteins, e.g., expression constructs containing two scfvs with a hydrophilic helical peptide linker between the two scfvs and the intact constant region, e.g., as described in US 5637481; multivalent and multispecific binding proteins, e.g., dimers of polypeptides having a first domain together with a binding region for an Ig heavy chain variable region and a second domain together with a binding region for an Ig light chain variable region, collectively referred to as diabodies (also encompassing higher order structures that produce bispecific, trispecific, or tetraspecific molecules), e.g., 2 as described in US 583724; a VL chain and a VH chain linked to a mini-antibody construct, said VL and VH chains also being linked to an antibody hinge region and a CH3 region by means of a peptide spacer sequence, which mini-antibody construct can dimerise to form a bispecific/multivalent molecule, for example as described in US 5837821; a VH domain and a VL domain linked in either orientation to a short peptide linker (e.g., 5 or 10 amino acids), or no linker at all, which can form a dimer to form a bispecific diabody; trimers and tetramers, for example, as described in US 5844094; a series of VH domains (or VL domains in family members) linked at the C-terminus by peptide bonds with a cross-linkable group, said domains being further associated with the VL domains to form a series of FVs (or scfvs), for example as described in US 5864019; and single-chain binding polypeptides in which both the VH domain and VL domain are linked by a peptide linker are incorporated into multivalent structures by means of non-covalent or chemical cross-linking to form, for example, homo-bivalent, hetero-bivalent, trivalent and tetravalent structures using scFV or diabody-type formats, e.g., as described in US 5869620. Additional exemplary multispecific and bispecific molecules and methods of producing these molecules are found, for example, in the following applications: US 5910573, US 5932448, US 5959083, US 5989830, US 6005079, US 6239259, US 6294353, US 6333396, US 6476198, US 6511663, US 6670453, US 6743896, US 6809185, US 6833441, US 7129330, US 7183076, US 7521056, US 7527787, US 7534866, US 7612181 a 7612181, US 7612181 a 36, US 2008171855 a1, US 2008241884 a1, US 2008254512 a1, US 2008260738 a1, US 2009130106 a1, US 2009148905 a1, US 2009155275 a1, US 2009162359 a1, US 2009162360 a1, US 2009175851 a1, US 2009175867 a1, US 2009232811 a1, US 2009234105 a1, US 2009263392 a1, US 2009274649 a1, EP 346087a2, WO 0006605 a2, WO 2 a2, WO 6372 a2, WO 2 a2, WO 2 a. The contents of the above-mentioned applications are incorporated herein by reference in their entirety.

Within each antibody or antibody fragment (e.g., scFv) of the bispecific antibody molecule, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is in accordance with its VH (VH)₁) At its VL (VL)₁) Upstream arranged and downstream antibodies or antibody fragments (e.g., scFv) according to their VL (VL)₂) In its VH (VH)₂) Arranged upstream such that the overall bispecific antibody molecule has the layout VH₁-VL₁-VL₂-VH₂. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is according to its VL (VL)₁) In its VH (VH)₁) Arranged upstream and downstream antibodies or antibody fragments (e.g., scFv) according to their VH (VH)₂) At its VL (VL)₂) Arranged upstream such that the overall bispecific antibody molecule has the layout VL₁-VH₁-VH₂-VL₂. Optionally, is connected toThe head is arranged between two antibodies or antibody fragments (e.g., scFv), e.g., if the construct is arranged as a VH₁-VL₁-VL₂-VH₂Is then arranged at VL₁And VL₂Or if the construct is arranged as a VL₁-VH₁-VH₂-VL₂Is then arranged in VH₁And VH₂In the meantime. The linker may be a linker as described herein, e.g., (Gly)₄-Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 4(SEQ ID NO: 26). In general, the linker between the two scfvs should be long enough to avoid mis-pairing between the domains of the two scfvs. Optionally, a linker is disposed between the VL and VH of the first scFv. Optionally, a linker is disposed between the VL and VH of the second scFv. In constructs having multiple linkers, any two or more linkers can be the same or different. Thus, in some embodiments, a bispecific CAR comprises a VL, a VH, and optionally one or more linkers in a layout as described herein.

In one aspect, the bispecific antibody molecule features a first immunoglobulin variable domain sequence (e.g., an scFv) having binding specificity for CD33, e.g., comprising an scFv as described herein (e.g., as described in table 2 or table 9), or comprising a light chain CDR and/or a heavy chain CDR from a CD33scFv as described herein, and a second immunoglobulin variable domain sequence having binding specificity for a second epitope on a different antigen. In some aspects, the second immunoglobulin variable domain sequence has binding specificity for an antigen expressed on AML cells (e.g., an antigen other than CD 33). For example, the second immunoglobulin variable domain sequence has binding specificity for CD 123. As another example, the second immunoglobulin variable domain sequence has binding specificity for CLL-1. As another example, the second immunoglobulin variable domain sequence has binding specificity for CD 34. As another example, the second immunoglobulin variable domain sequence has binding specificity for FLT 3. For example, the second immunoglobulin variable domain sequence has binding specificity for folate receptor beta. In some aspects, the second immunoglobulin variable domain sequence has binding specificity for an antigen expressed on a B cell (e.g., CD19, CD20, CD22, or ROR 1).

Chimeric TCR

In one aspect, the CD33 antibodies and antibody fragments of the invention (e.g., those disclosed in tables 2 and 9) can be grafted to one or more constant domains of a T cell receptor ("TCR") chain (e.g., a TCR α or TCR β chain) to generate a chimeric TCR that specifically binds to CD 33. Without being bound by theory, it is believed that the chimeric TCR will signal through the TCR complex once bound to the antigen. For example, a CD33scFv as disclosed herein can be grafted to a constant domain, e.g., at least a portion of an extracellular constant domain, transmembrane domain, and cytoplasmic domain of a TCR chain (e.g., a TCR a chain and/or a TCR β chain). As another example, a CD33 antibody fragment, e.g., a VL domain as described herein, can be grafted to a constant domain of a TCR α chain, and a CD33 antibody fragment, e.g., a VH domain as described herein, can be grafted to a constant domain of a TCR β chain (or alternatively, a VL domain can be grafted to a constant domain of a TCR β chain and a VH domain can be grafted to a TCR α chain). As another example, the CDRs of a CD33 antibody or antibody fragment (e.g., the CDRs of a CD33 antibody or antibody fragment as described in table 3, table 4, table 10, table 11, table 12, or table 13) can be grafted into TCR α and/or β chains to generate a chimeric TCR that specifically binds to CD 33. For example, the LCDRs disclosed herein may be grafted into the variable domain of the TCR α chain and the HCDRs disclosed herein may be grafted into the variable domain of the TCR β chain, or vice versa. Such chimeric TCRs can be generated by methods known in the art (e.g., Willemsen RA et al, Gene Therapy 2000; 7: 1369-.

Transmembrane domain

With respect to the transmembrane domain, in various embodiments, the CAR can be designed to comprise a transmembrane domain linked to the extracellular domain of the CAR. The transmembrane domain may include one or more additional amino acids contiguous with the transmembrane region, e.g., one or more amino acids associated with an extracellular region of a protein from which the transmembrane region is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 extracellular region amino acids) and/or one or more amino acids associated with an intracellular region of a protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 intracellular region amino acids). In one aspect, the transmembrane domain is a domain that associates with one of the other domains of the CAR used. In some cases, the transmembrane domains may be selected or modified by amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other components of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerizing with another CAR on the surface of a CAR-expressing cell (e.g., a CART cell). In a different aspect, the amino acid sequence of the transmembrane domain can be modified or replaced to minimize interaction with the binding domain of a native binding partner present in the same cell that expresses the CAR (e.g., CART).

The transmembrane domain may be derived from a natural source or a recombinant source. Where the source is native, the domain may be derived from any membrane bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling the intracellular domain whenever the CAR has bound to the target. Transmembrane domains particularly useful in the present invention may comprise, for example, the transmembrane domains of the following proteins: the α, β, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 α, CD8 β), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154. In some embodiments, the transmembrane domain may include at least the transmembrane regions of, for example, the following proteins: KIRDS, OX, CD, LFA-1(CD11, CD), ICOS (CD278), 4-1BB (CD137), GITR, CD, BAFFR, HVEM (LIGHT TR), SLAMF, NKp (KLRF), NKp, CD160, CD, IL2 β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, LFA-1, ITGAX, CD11, ITGB, CD, LFA-1, ITGB, TNFR, TNPM (CD226), SLAMF (CD244, 2B), CD (Talle), CEACAM, TAM, LyGB, GAM (CD229), CD160, SLPAG (SLPAG), SLAMG-100, SLAMF (SLAMF), SLAMF (SLAMBR-150), SLAMBR-2B, SLAGE-2, SLAGE-2, TAMB, TAM, CD229, TAMB, TARG.

In some cases, the transmembrane domain can be linked to the extracellular region of the CAR (e.g., the antigen binding domain of the CAR) via a hinge (e.g., a hinge from a human protein). For example, in one embodiment, the hinge may be a human Ig (immunoglobulin) hinge, e.g., an IgG4 hinge region, or a CD8 hinge region. In one embodiment, the hinge or spacer sequence comprises (e.g., consists of) the amino acid sequence of SEQ ID NO: 2. In one aspect, the transmembrane domain comprises (e.g., consists of) the transmembrane domain of SEQ ID NO 6.

In one aspect, the hinge or spacer sequence comprises an IgG4 hinge. For example, in one embodiment, the hinge or spacer sequence comprises a hinge having the amino acid sequence:

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO: 3). In some embodiments, the hinge or spacer sequence comprises a hinge encoded by the nucleotide sequence:

GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG(SEQ ID NO:14)。

in one aspect, the hinge or spacer sequence comprises an IgD hinge. For example, in one embodiment, the hinge or spacer sequence comprises a hinge having the amino acid sequence:

RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO: 4). In some embodiments, the hinge or spacer sequence comprises a hinge encoded by the nucleotide sequence:

AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT(SEQ ID NO:15)。

in one aspect, the transmembrane domain may be recombinant, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan, and valine can be present at each end of the recombinant transmembrane domain.

Optionally, a short oligopeptide linker or polypeptide linker between 2 and 10 amino acids in length can form a linkage between the transmembrane domain and the cytoplasmic domain of the CAR. The glycine-serine doublet provides a particularly suitable linker. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 5). In some embodiments, the linker is encoded by the nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 16).

In one aspect, the hinge or spacer sequence comprises a KIR2DS2 hinge.

Cytoplasmic Domain

The cytoplasmic domain or region of the CAR of the invention includes an intracellular signaling domain. The intracellular signaling domain is capable of activating at least one normal effector function of an immune cell into which the CAR has been introduced.

Examples of intracellular signaling domains for use in the CARs of the invention include cytoplasmic sequences of the T Cell Receptor (TCR) and co-receptor that act synergistically to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence with the same functional capability.

It is known that the signal generated by the TCR alone is not sufficient to fully activate T cells and that secondary and/or co-stimulatory signals are also required. Thus, T cell activation is said to be mediated by two distinct classes of cytoplasmic signaling sequences: those sequences that initiate antigen-dependent primary activation by the TCR (primary intracellular signaling domains) and those that function in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains).

The primary signaling domain modulates primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary intracellular signaling domain, which functions in a stimulatory manner, may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

Examples of primary intracellular signaling sequences containing ITAMs that are particularly useful in the present invention include those of TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), fcε RI, DAP10, DAP12, and CD66 d. In one embodiment, the CAR of the invention comprises an intracellular signaling domain, e.g., the primary signaling domain of CD 3-zeta.

In one embodiment, the primary signaling domain comprises a modified ITAM domain, e.g., a mutant ITAM domain having altered (e.g., increased or decreased) activity as compared to a native ITAM domain. In one embodiment, the primary signaling domain comprises a primary intracellular signaling domain comprising a modified ITAM, e.g., a primary intracellular signaling domain comprising an optimized and/or truncated ITAM. In one embodiment, the primary signaling domain comprises one, two, three, four or more ITAM motifs.

Other examples of molecules containing primary intracellular signaling domains that are particularly useful in the present invention include those of DAP10, DAP12, and CD 32.

The intracellular signaling domain of a CAR may itself comprise a primary signaling domain, e.g., the CD 3-zeta signaling domain, or it may be combined with any other desired intracellular signaling domain useful in the context of the CARs of the invention. For example, the intracellular signaling domain of a CAR can comprise a primary signaling domain (e.g., a CD 3-zeta chain portion) and a costimulatory signaling domain. Costimulatory signaling domain refers to the portion of the CAR that comprises the intracellular domain of the costimulatory molecule. Costimulatory molecules are cell surface molecules other than the antigen receptor or its ligand that are required by lymphocytes to respond effectively to an antigen. Examples of such molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1(CD11a/CD18), 4-1BB (CD137), B7-H7, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHT), KIRDS 7, SLAMF7, NKp 7 (KLRF 7), NKp 7, CD7 alpha, CD7 beta, IL 27 gamma, VLITalpha 7, VLITGA 7, GAITGB 11, GAITGA 7, GAITGB 11-7, GAITGB 11, GAITGA 7, GAITGB 7, GAIT11, GAITGA 7, GAITGB 7, GAITGA 7, GAITGB 11, GAIT11, GAITGA 7, GAITGB 7, GAIT11-7, GAITGB 7, GAIT11-7, GAITGB 7, GAIT11, GAITGA 7, GAITGB 7, GAIT11-7, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and ligands that bind specifically to CD 83. For example, CD27 co-stimulation has been demonstrated to enhance expansion, effector function and survival of human CART cells in vitro and to enhance human T cell retention and anti-tumor activity in vivo (Song et al, blood.2012; 119(3): 696-706).

Intracellular signaling sequences within the cytoplasmic portion of the CAR of the invention can be linked to each other in random order or in a designated order. Optionally, short oligopeptide linkers or polypeptide linkers, e.g., between 2 and 10 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), can form a linkage between intracellular signaling sequences. In one embodiment, a glycine-serine doublet may be used as a suitable linker. In one embodiment, a single amino acid, e.g., alanine, glycine, may be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed to comprise two or more (e.g., 2, 3, 4, 5, or more) costimulatory signaling domains. In one embodiment, two or more (e.g., 2, 3, 4, 5, or more) co-stimulatory signaling domains are separated by a linker molecule (e.g., a linker molecule described herein). In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD 3-zeta and the signaling domain of CD 28. In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD 3-zeta and the signaling domain of 4-1 BB. In one aspect, the signaling domain of 4-1BB is the signaling domain of SEQ ID NO. 7. In one aspect, the signal domain of CD 3-zeta is the signal domain of SEQ ID NO 9 (mutant CD 3-zeta) or SEQ ID NO 10 (wild type human CD 3-zeta).

In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD 3-zeta and the signaling domain of CD 27. In one aspect, the signal domain of CD27 comprises the amino acid sequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 8).

In one aspect, the signal domain of CD27 is encoded by the nucleic acid sequence of AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 19).

In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD 3-zeta and the signaling domain of CD 28. In one aspect, the signal domain of CD28 comprises the amino acid sequence of SEQ ID NO: 379. In one aspect, the signal domain of CD28 is encoded by the nucleic acid sequence of SEQ ID NO. 380.

In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD 3-zeta and the signaling domain of ICOS. In one aspect, the signal domain of CD28 comprises the amino acid sequence of SEQ ID NO 381. In one aspect, the signal domain of ICOS is encoded by the nucleic acid sequence of SEQ ID NO: 382.

In one aspect, a CAR-expressing cell described herein can further comprise a second CAR, e.g., a second CAR comprising a different antigen binding domain, e.g., to the same target (CD33) or to a different target (e.g., CD123, CLL-1, CD34, FLT3, or folate receptor β). In one embodiment, the second CAR comprises an antigen binding domain against a target expressed on acute myeloid leukemia cells (e.g., CD123, CLL-1, CD34, FLT3, or folate receptor beta). In one embodiment, the CAR-expressing cell comprises a first CAR that targets a first antigen and includes an intracellular signaling domain with a costimulatory signaling domain but no primary signaling domain, and a second CAR that targets a second, different antigen and includes an intracellular signaling domain with a primary signaling domain but no costimulatory signaling domain. While not wishing to be bound by theory, placement of a costimulatory signaling domain (e.g., 4-1BB, CD28, CD27, ICOS, or OX-40) onto a first CAR, and placement of a primary signaling domain (e.g., CD3 ζ) onto a second CAR can limit the activity of the CARs to cells expressing both targets. In one embodiment, a cell expressing a CAR comprises a first CD33 CAR comprising a CD33 binding domain, a transmembrane domain, and a costimulatory domain, and a second CAR that targets an antigen other than CD33 (e.g., an antigen expressed on AML cells, e.g., CD123, CLL-1, CD34, FLT3, or folate receptor beta) and comprises an antigen binding domain, a transmembrane domain, and a primary signal domain. In another embodiment, a cell expressing a CAR comprises a first CD33 CAR comprising a CD33 binding domain, a transmembrane domain, and a primary signal domain, and a second CAR that targets an antigen other than CD33 (e.g., an antigen expressed on AML cells, e.g., CD123, CLL-1, CD34, FLT3, or folate receptor beta) and comprises an antigen binding domain, a transmembrane domain, and a costimulatory signal domain for the antigen.

In one embodiment, the CAR-expressing cell comprises a CD33 CAR and an inhibitory CAR described herein. In one embodiment, the inhibitory CAR comprises an antigen binding domain that binds to an antigen that is present on normal cells, but not on cancer cells, e.g., normal cells that also express CD 33. In one embodiment, the inhibitory CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. For example, the intracellular domain of an inhibitory CAR may be an intracellular domain of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR β.

In one embodiment, when the CAR-expressing cell comprises two or more different CARs, the antigen-binding domains of the different CARs may be such that the antigen-binding domains do not interact with each other. For example, a cell expressing the first and second CARs may have the antigen binding domain of the first CAR, e.g., as a fragment (e.g., scFv) that does not form an association with the antigen binding domain of the second CAR, e.g., the antigen binding domain of the second CAR is VHH.

In some embodiments, the antigen binding domain comprises a Single Domain Antigen Binding (SDAB) molecule, including molecules whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, binding molecules that naturally lack a light chain, single domains derived from conventional four-chain antibodies, engineered domains, and single domain scaffolds other than those derived from antibodies. The SDAB molecule may be any molecule of the prior art, or any single domain molecule in the future. SDAB molecules may be derived from any species, including but not limited to mouse, human, camel, alpaca, lamprey, fish, shark, goat, rabbit, and cow. This term also includes naturally occurring single domain antibody molecules from species other than camelids (camelids) and sharks.

In one aspect, the SDAB molecule may be derived from the variable region of an immunoglobulin present in fish, such as, for example, that derived from an immunoglobulin isotype known as neoantigen receptor (NAR) present in shark serum. Methods for generating single domain molecules ("IgNARs") derived from the NAR variable region are described in WO 03/014161 and Streltsov (2005) Protein Sci.14: 2901-2909.

According to another aspect, the SDAB molecule is a naturally occurring single domain antigen binding molecule, referred to as a heavy chain lacking a light chain. Such single domain molecules are disclosed, for example, in WO 9404678 and Hamers-Casterman, C. et al, (1993) Nature 363: 446-. For clarity reasons, such variable domains derived from heavy chain molecules that naturally lack a light chain are referred to herein as VHHs or nanobodies to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules may be derived from camelus (Camelidae) species, such as camel, alpaca, dromedary, camel and guanaco. Other species than camelids may produce heavy chain molecules that naturally lack a light chain; such VHHs are within the scope of the invention.

SDAB molecules can be recombinant, CDR grafted, humanized, camelized, de-immunized, and/or generated in vitro (e.g., by phage display selection).

It has also been found that cells having multiple membrane-embedded chimeric receptors that contain antigen-binding domains that interact between the antigen-binding domains of the receptors may be undesirable, for example, because of its ability to inhibit the binding of one or more antigen-binding domains to their respective antigens. Thus, disclosed herein are cells having first and second non-naturally occurring membrane-embedded chimeric receptors comprising an antigen binding domain that minimizes such interactions. Also disclosed herein are nucleic acids encoding first and second non-naturally occurring membrane-embedded chimeric receptors comprising an antigen binding domain that minimizes such interactions. And methods of making and using such cells and nucleic acids. In one embodiment, the antigen binding domain of one of the first and the second non-naturally occurring membrane-embedded chimeric receptors comprises an scFv and the other comprises a single VH domain, e.g., a camelid, shark or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.

In some embodiments, the claimed invention comprises a first and a second CAR, wherein the antigen binding domain of one of the first CAR and the second CAR does not comprise a variable light chain domain and a variable heavy chain domain. In some embodiments, the antigen binding domain of one of the first CAR and the second CAR is an scFv and the other is not an scFv. In some embodiments, the antigen binding domain of one of the first CAR and the second CAR comprises a single VH domain, e.g., a camelid, shark or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen binding domain of one of the first CAR and the second CAR comprises a nanobody. In some embodiments, the antigen binding domain of one of the first CAR and the second CAR comprises a camelid VHH domain.

In some embodiments, the antigen binding domain of one of the first CAR and the second CAR comprises a scFv and the other comprises a single VH domain, e.g., a camelid, shark or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen binding domain of one of the first CAR and the second CAR comprises an scFv and the other comprises a nanobody. In some embodiments, the antigen binding domain of one of the first CAR and the second CAR comprises an scFv and the other comprises a camelid VHH domain.

In some embodiments, the binding of the antigen binding domain of the first CAR to its corresponding antigen is not substantially reduced by the presence of the second CAR when present on the surface of a cell. In some embodiments, the binding of the antigen binding domain of the first CAR to its corresponding antigen in the presence of the second CAR is 85%, 90%, 95%, 96%, 97%, 98% or 99% of the binding of the antigen binding domain of the first CAR to its corresponding antigen in the absence of the second CAR.

In some embodiments, the antigen binding domain of the first CAR and the antigen binding domain of the second CAR, when present on the surface of a cell, associate less with each other than when both antigen binding domains are scFv antigen binding domains. In some embodiments, the antigen binding domain of the first CAR and the antigen binding domain of the second CAR associate 85%, 90%, 95%, 96%, 97%, 98%, or 99% less with each other than if both antigen binding domains were scFv antigen binding domains.

In another aspect, a CAR-expressing cell described herein can also express another agent, e.g., an agent that enhances the activity of the CAR-expressing cell. For example, in one embodiment, the substance may be a substance that inhibits inhibitory molecules. In some embodiments, the inhibitory molecule (e.g., PD1) may reduce the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR β. In one embodiment, the agent that inhibits an inhibitory molecule is, e.g., a molecule described herein, e.g., an agent comprising a first polypeptide (e.g., an inhibitory molecule) associated with a second polypeptide (e.g., an intracellular signaling domain described herein) that provides a positive signal to a cell. In one embodiment, the agent comprises a first polypeptide, e.g., an inhibitory molecule such as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2 ζ 29, MHC class I, MHC class II, GAL9, adenosine, and TGFR β, or a fragment of any of these molecules (e.g., at least a portion of the ectodomain of any of these molecules) and an intracellular signaling domain as described herein (e.g., a signaling domain comprising a co-stimulatory domain (e.g., CD 41, BB 4642, CD 4642 as described herein) and a second signaling domain (e.g., a signaling domain of the embodiments described herein, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein). In embodiments, the CAR-expressing cells described herein comprise a switch co-stimulatory receptor, e.g., as described in WO2013/019615, which is incorporated herein in its entirety by reference. PD1 is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al, 1996int. Immunol 8: 765-75). Two ligands for PD1, PD-L1 and PD-L2 have been shown to down-regulate T cell activation when bound to PD1 (Freeman et al, 2000J Exp Med 192: 1027-34; Latchman et al, 2001 Nat Immunol 2: 261-8; Carter et al, 2002Eur J Immunol 32: 634-43). PD-L1 is abundant in human cancers (Dong et al, 2003J Mol Med 81: 281-7; Blank et al, 2005 Cancer Immunol. Immunother 54: 307-314; Konishi et al, 2004 Clin Cancer Res 10: 5094). Immunosuppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.

In one embodiment, the agent comprises an extracellular domain (ECD) of an inhibitory molecule (e.g., programmed death 1(PD1)) that can be fused to a transmembrane domain and an intracellular signaling domain such as 41BB and CD3 ζ (also referred to herein as PD1 CAR). In one embodiment, the PD1 CAR improves the persistence of a cell (e.g., a T cell or NK cell) expressing the CAR when used in combination with a CD33 CAR described herein. In one embodiment, the CAR is a PD1 CAR comprising the PD1 extracellular domain as shown underlined in SEQ ID NO: 24. In one embodiment, the PD1 CAR comprises the amino acid sequence of SEQ ID NO. 24.

Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnw yrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelr vterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr(SEQ ID NO:24)。

In one embodiment, the PD1 CAR comprises the amino acid sequence provided below (SEQ ID NO: 22).

pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsq pgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpag qfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr(SEQ ID NO:22)。

In one embodiment, the agent comprises a nucleic acid sequence encoding a PD1 CAR (e.g., a PD1 CAR described herein). In one embodiment, the nucleic acid sequence of PD1 CAR is shown below, and PD1ECD is underlined in SEQ ID NO:23, below

atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagaccacccgg atggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggc gataatgcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgt caaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgac tcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgc ggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagag ctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaagcggggtcggaaaaagcttctgtacattttcaagcagcccttcatgaggcccgtgcaaaccacccaggaggaggacggttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacgatgccctgcacatgcaggcccttccccctcgc(SEQ ID NO:23)。

In another aspect, the invention provides a population of CAR-expressing cells (e.g., CART cells or CAR-expressing NK cells). In some embodiments, the population of cells expressing a CAR comprises a mixture of cells expressing different CARs. For example, in one embodiment, a population of CAR-expressing cells (e.g., CART cells or CAR-expressing NK cells) can comprise a first cell expressing a CAR having a CD33 binding domain described herein and a second cell expressing a CAR having a different CD33 binding domain (e.g., a CD33 binding domain described herein that is different from the CD33 binding domain in the CAR expressed by the first cell). As another example, a population of cells expressing a CAR can include a first cell expressing a CAR that includes a CD33 binding domain (e.g., as described herein), and a second cell expressing a CAR that includes an antigen binding domain against a target other than CD33 (e.g., CD123, CD34, CLL-1, FLT3, or folate receptor beta). In one embodiment, the population of cells expressing a CAR comprises, e.g., a first cell expressing a CAR comprising a primary intracellular signaling domain, and a second cell expressing a CAR comprising a secondary signaling domain, e.g., a costimulatory signaling domain.

In another aspect, the invention provides a population of cells, wherein at least one cell in the population expresses a CAR having a CD33 binding domain as described herein, and a second cell that expresses another agent (e.g., an agent that enhances the activity of the CAR-expressing cell). For example, in one embodiment, the substance may be a substance that inhibits inhibitory molecules. In some embodiments, the inhibitory molecule, for example, can reduce the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR β. In one embodiment, for example, described herein are agents that inhibit an inhibitory molecule, comprising a first polypeptide (e.g., an inhibitory molecule) associated with a second polypeptide that provides a positive signal to a cell (e.g., an intracellular signaling domain described herein). In one embodiment, the agent comprises a first polypeptide, e.g., an inhibitory molecule such as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2 ζ 29, MHC class I, MHC class II, GAL9, adenosine, and TGFR β, or a fragment of any of these molecules (e.g., at least a portion of the ectodomain of any of these molecules) and an intracellular signaling domain as described herein (e.g., a signaling domain comprising a co-stimulatory domain (e.g., CD 41, BB 4642, CD 4642 as described herein) and a second signaling domain (e.g., a signaling domain of the embodiments described herein, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD 1) and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein).

In one aspect, the invention provides methods comprising administering a population of CAR-expressing cells (e.g., CART cells or CAR-expressing NK cells), e.g., a mixture of cells expressing different CARs, in combination with another agent (e.g., a kinase inhibitor, such as a kinase inhibitor described herein). In another aspect, the invention provides methods of administering a population of cells in combination with another agent (e.g., a kinase inhibitor, such as a kinase inhibitor described herein), wherein at least one cell in the population expresses a CAR having an anti-cancer associated antigen-binding domain as described herein, and a second cell that expresses another agent (e.g., an agent that enhances the activity of the CAR-expressing cell).

Natural killer cell receptor (NKR) CAR

In one embodiment, the CAR molecules described herein comprise one or more components of a natural killer cell receptor (NKR), thus forming an NKR-CAR. The NKR component may be a transmembrane, hinge or cytoplasmic domain from any of the following natural killer cell receptors: killer immunoglobulin-like receptors (KIRs), such as KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP 1; natural Cytotoxic Receptors (NCRs), e.g., NKp30, NKp44, NKp 46; the Signaling Lymphocyte Activating Molecule (SLAM) family of immune cell receptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRA, BLAME, and CD 2F-10; fc receptors (fcrs), e.g., CD16, and CD 64; and Ly49 receptors, e.g., Ly49A, Ly 49C. The NKR-CAR molecules described herein can interact with an adaptor molecule or an intracellular signaling domain (e.g., DAP 12). Exemplary layouts and sequences of CAR molecules comprising NKR components are described in international publication No. WO2014/145252, the contents of which are thus incorporated by reference in their entirety.

Strategies for modulating chimeric antigen receptors

There are many ways in which CAR activity can be modulated. In some embodiments, a modulatable CAR (rcar) in which CAR activity can be controlled is one that optimizes the safety and efficacy needs of the CAR therapy. For example, induction of apoptosis using, for example, a caspase fused to the dimerization domain (see, e.g., Di et al, N Engl. J. Med.2011 Nov. 3; 365(18): 1673-. In another example, when a dimerization drug that causes caspase-9 activation and apoptosis (e.g., rimiducid (also known as AP1903(Bellicum Pharmaceuticals) or AP20187(Ariad)) is administered, cells expressing the CAR may also express an inducible caspase-9 (iCaspase-9) molecule, the iCaspase-9 molecule contains a dimerization process Chemical Inducer (CID) domain that mediates dimerization in the presence of CID, which results in inducible and selective depletion of cells expressing the CAR. N.engl.j.med.2011; 365:1673-83.

Alternative strategies to modulate the CAR therapy of the invention include the use of small molecules or antibodies that inactivate or turn off CAR activity, e.g., by eliminating cells expressing the CAR, e.g., by inducing antibody-dependent cell-mediated cytotoxicity (ADCC). For example, a CAR-expressing cell described herein can also express an antigen recognized by a molecule capable of inducing cell death (e.g., ADCC or complement-induced cell death). For example, the CAR-expressing cells described herein can also express a receptor that can be targeted by the antibody or antibody fragment. Examples of such receptors include EPCAM, VEGFR, integrins (e.g., integrins α v β 3, α 4α Ι β 3, α 4 β 7, α 5 β 1, α v β 3, α v), TNF receptor superfamily members (e.g., TRAIL-R), PDGF receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC, TAG-72, IL-6 receptor, 5T, GD, GDB, CD 11/LFA-1, CD/ITGB, CD/lgE receptor, CD62, CD125, CD 147/design, CD152/CTLA-4, CD154/CD40, CD195/CCR, CD319/SLAMF, and EGFR truncations and forms thereof (e.g., forms that retain one or more extracellular epitopes but one or more cytoplasmic domains). For example, the CAR-expressing cells described herein may also express a truncated Epidermal Growth Factor Receptor (EGFR) that lacks signaling capability, but retains ADCC-inducing molecules (e.g., cetuximab)

) The epitope recognized, whereby administration of cetuximab results in ADCC and subsequent depletion of CAR-expressing cells (see, e.g., WO 2011/056894, and jonnagaadd et al, Gene ther.2013; 20(8)853-860). Another strategy involves expressing a highly compact marker/suicide gene that incorporates target epitopes from both the CD32 antigen and the CD20 antigen in CAR-expressing cells described herein, which highly compact marker/suicide gene expression binds rituximab resulting in selective depletion of CAR-expressing cells, e.g., by ADCC (see, e.g., Philip et al, blood.2014; 124(8) 1277-. Other methods for depleting the CAR-expressing cells described herein include administering

A monoclonal anti-CD 52 antibody that selectively binds to and targets mature lymphocytes (e.g., CAR-expressing cells) for destruction (e.g., by inducing ADCC). In other embodiments, a CAR ligand (e.g., an anti-idiotype antibody) can be used to selectively target a CAR-expressing cell. In some embodimentsThe anti-idiotype antibody may cause effector cell activity, such as ADCC or ADC activity, thereby reducing the number of cells expressing the CAR. In other embodiments, a CAR ligand, e.g., an anti-idiotype antibody, can be coupled to a substance (e.g., a toxin) that induces cell killing, thereby reducing the number of cells expressing the CAR. Alternatively, the CAR molecule itself can be configured such that activity, e.g., on and off, can be modulated, as described below.

In some embodiments, the RCAR comprises a set of polypeptides, typically two polypeptides in the simplest embodiment, wherein the components of a standard CAR described herein (e.g., an antigen binding domain and an intracellular signaling domain) are distributed over separate polypeptides or building blocks. In some embodiments, the set of polypeptides includes a dimerization switch, wherein the dimerization switch can couple the polypeptides to each other, e.g., can couple an antigen binding domain to an intracellular signaling domain, in the presence of a dimerization molecule. Additional descriptions and exemplary layouts of such regulated CARs are provided herein and in international publication number WO 2015/090229, which is hereby incorporated by reference in its entirety.

In one embodiment, the RCAR comprises two polypeptides or components: 1) an intracellular signaling component comprising an intracellular signaling domain (e.g., a primary intracellular signaling domain described herein) and a first switching domain; 2) an antigen binding member comprising an antigen binding domain as described herein (e.g., targeted to a tumor antigen described herein) and a second switch domain. Optionally, the RCAR comprises a transmembrane domain as described herein. In one embodiment, the transmembrane domain may be provided on the intracellular signaling component, on the antigen binding component, or on both. (unless otherwise indicated, the order in which components or elements of an RCAR are described herein may be as provided, but other orders may also be included). In other words, in one embodiment, the order is as described in the text, but in other embodiments, the order may be different. For example, the order of elements on one side of the transmembrane region may differ from the examples, e.g., the placement of the switching domain relative to the intracellular signaling domain may be different, e.g., reversed.

In one embodiment, the first switch domain and the second switch domain may form an intracellular or extracellular dimerization switch. In one embodiment, the dimerization switch may be a homodimerization switch, e.g., wherein the first switch domain and the second switch domain are the same, or a heterodimerization switch, e.g., wherein the first switch domain and the second switch domain are different from each other.

In an embodiment, the RCAR may comprise a "multiple switch. The multiple switch may comprise a heterodimeric switch domain or a homodimeric switch domain. A multiplex switch independently comprises a plurality, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 switch domains on a first building block (e.g., an antigen binding building block) and a second building block (e.g., an intracellular signaling building block). In one embodiment, the first building block may comprise a plurality of first switch domains, e.g., FKBP-based switch domains, and the second building block may comprise a plurality of second switch domains, e.g., FRB-based switch domains. In one embodiment, the first building block may comprise a first switching domain and a second switching domain, e.g., an FKBP-based switching domain and an FRB-based switching domain, and the second building block may comprise a first switching domain and a second switching domain, e.g., an FKBP-based switching domain and an FRB-based switching domain.

In one embodiment, the intracellular signaling component comprises one or more intracellular signaling domains (e.g., a primary intracellular signaling domain and one or more costimulatory signaling domains).

In one embodiment, the antigen binding member may comprise one or more intracellular signaling domains, e.g., one or more costimulatory signaling domains. In one embodiment, the antigen binding member comprises a plurality, e.g., 2 or 3, of the costimulatory signaling domains described herein, e.g., a costimulatory signaling domain selected from the group consisting of 4-1BB, CD28, CD27, ICOS, and OX40, and in various embodiments, does not comprise a primary intracellular signaling domain. In one embodiment, the antigen binding member comprises the following co-stimulatory signaling domains in the extracellular to intracellular direction: 4-1BB-CD 27; 41BB-CD 27; CD27-4-1 BB; 4-1BB-CD 28; CD28-4-1 BB; OX40-CD 28; CD28-OX 40; CD28-4-1 BB; or 4-1BB-CD 28. In such embodiments, the intracellular binding member comprises a CD3 zeta domain. In such an embodiment, the RCAR comprises (1) an antigen binding member comprising an antigen binding domain, a transmembrane domain, and two costimulatory domains and a first switch domain; and (2) an intracellular signaling domain comprising a transmembrane domain or a membrane-binding domain and at least one primary intracellular signaling domain and a second switch domain.

One embodiment provides the RCAR wherein the antigen binding member is not tethered to the cell surface of the CAR. This allows cells with intracellular signaling constructs to be conveniently paired with one or more antigen binding domains without transforming the cells with sequences encoding the antigen binding constructs. In such embodiments, the RCAR comprises: 1) an intracellular signaling component comprising: a first switch domain, a transmembrane domain, an intracellular signaling domain (e.g., a primary intracellular signaling domain), and a first switch domain; and 2) an antigen binding member comprising: an antigen binding domain and a second switch domain, wherein the antigen binding member does not comprise a transmembrane domain or a membrane-binding domain and optionally does not comprise an intracellular signaling domain. In some embodiments, the RCAR may further comprise 3) a second antigen-binding member comprising a second antigen-binding domain, e.g., a second antigen-binding domain that binds a different antigen bound by the antigen-binding domain; and a second switch domain.

Also provided herein are RCARs wherein the antigen binding member comprises bispecific activation and targeting capabilities. In this embodiment, the antigen binding member may comprise a plurality, e.g., 2, 3, 4, or 5 antigen binding domains, e.g., scfvs, wherein each antigen binding domain binds to a target antigen, e.g., a different antigen or the same antigen, e.g., the same or different epitopes on the same antigen. In one embodiment, a plurality of antigen binding domains are in tandem, and optionally a linker or hinge region is disposed between each antigen binding domain. Suitable linkers and hinge regions are described herein.

One embodiment provides an RCAR having a layout that allows for proliferation transformations. In this embodiment, the RCAR comprises: 1) an intracellular signaling component comprising: optionally, a transmembrane domain or a membrane-binding domain; one or more costimulatory signal domains (e.g., selected from the group consisting of 4-1BB, CD28, CD27, ICOS, and OX 40), and a switch domain; and 2) an antigen binding member comprising: an antigen binding domain, a transmembrane domain, and a primary intracellular signaling domain (e.g., a CD3 zeta domain), wherein the antigen binding member does not comprise a switch domain or does not comprise a switch domain that dimerizes with a switch domain on an intracellular signaling member. In one embodiment, the antigen binding member does not comprise a costimulatory signal domain. In one embodiment, the intracellular signaling member comprises a switch domain from a homodimerization switch. In one embodiment, the intracellular signaling member comprises a first switch domain of a heterodimerization switch, and the RCAR comprises a second intracellular signaling member comprising a second switch domain of a heterodimerization switch. In such embodiments, the second intracellular signaling component comprises the same intracellular signaling domain as the intracellular signaling component. In one embodiment, the dimerization switch is intracellular. In one embodiment, the dimerization switch is extracellular.

In any of the RCAR layouts described herein, the first and second switch domains comprise FKBP-FRB based switches as described herein.

Also provided herein are cells comprising the RCARs described herein. Any cell engineered to express RCAR may be used as an RCARX cell. In one embodiment, the RCARX cells are T cells and are referred to as RCART cells. In one embodiment, the RCARX cells are NK cells and are referred to as RCARN cells.

Also provided herein are nucleic acids and vectors comprising a sequence encoding an RCAR. The sequences encoding the various elements of the RCAR may be provided on the same nucleic acid molecule, e.g., the same plasmid or vector, e.g., a viral vector, e.g., a lentiviral vector. In one embodiment, (i) the sequence encoding the antigen binding member and (ii) the sequence encoding the intracellular signaling member may be present on the same nucleic acid (e.g., vector). Production of the corresponding protein may be achieved, for example, by using separate promoters, or by using a bicistronic transcription product (which may result in the production of both proteins, either by cleavage of a single translation product or by translation of two separate protein products). In one embodiment, a sequence encoding a cleavable peptide (e.g., a P2A or F2A sequence) is disposed between (i) and (ii). In one embodiment, the sequence encoding the IRES (e.g., EMCV or EV71IRES) is arranged between (i) and (ii). In these embodiments, (i) and (ii) are transcribed as a single RNA. In one embodiment, the first promoter is operably linked to (i) and the second promoter is operably linked to (ii), such that (i) and (ii) are transcribed as separate mrnas.

Alternatively, the sequences encoding the various elements of the RCAR may be disposed on different nucleic acid molecules, e.g., different plasmids or vectors, e.g., viral vectors, e.g., lentiviral vectors. For example, (i) a sequence encoding an antigen binding member may be present on a first nucleic acid (e.g., a first vector), and (ii) a sequence encoding an intracellular signaling member may be present on a second nucleic acid (e.g., a second vector).

Dimerization switch

The dimerization switch may be non-covalent or covalent. In non-covalent dimerization switches, dimerization molecules facilitate non-covalent interactions between switch domains. In covalent dimerization switches, dimerization molecules facilitate covalent interactions between switch domains.

In one embodiment, the RCAR comprises an FKBP/FRAP-based or FKBP/FRB-based dimerization switch. FKBP12(FKBP, or FK506 binding protein) is an abundant cytoplasmic protein that serves as the primary intracellular target of the natural product immunosuppressive drug rapamycin. Rapamycin binds to FKBP and to FRAP (RAFT, mTOR), a large PI3K homolog. FRB is the 93 amino acid portion of FRAP sufficient to bind the FKBP-rapamycin complex (Chen, J., ZHEN, X.F., Brown, E.J., and Schreiber, S.L. (1995) Identification of an 11-kDa FKBP12-rapamycin-binding domain with the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical server response. Proc Natl Acad Sci U A92: 4947-51).

In embodiments, FKBP/FRAP-based (e.g., FKBP/FRB-based) switches can use dimeric molecules, e.g., rapamycin or rapamycin analogs.

The amino acid sequence of FKBP is as follows:

D V P D Y A S L G G P S S P K K K R K V S R G V Q V E T I S P G D G R T F P K R G Q T C V V H Y T G M L E D G K K F D S S R D R N K P F K F M L G K Q E V I R G W E E G V A Q M S V G Q R A K L T I S P D Y A Y G A T G H P G I I P P H A T L V F D V E L L K L E T S Y(SEQ ID NO:148)。

in embodiments, the FKBP switch domain may comprise a FKBP fragment having the ability to bind to FRB or a fragment or analog thereof in the presence of rapamycin or a rapamycin analog, e.g., the underlined portion of SEQ ID NO:148 which is:

V Q V E T I S P G D G R T F P K R G Q T C V V H Y T G M L E D G K K F D S S R D R N K P F K F M L G K Q E V I R G W E E G V A Q M S V G Q R A K L T I S P D Y A Y G A T G H P G I I P P H A T L V F D V E L L K L E T S(SEQ ID NO:149)。

the amino acid sequence of FRB is as follows:

ILWHEMWHEG LEEASRLYFG ERNVKGMFEV LEPLHAMMER GPQTLKETSF NQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR ISK(SEQ ID NO:150)

as the term is used herein, a "FKBP/FRAP (e.g., FKBP/FRB based) switch" refers to a dimerization switch comprising a first switch domain comprising an FKBP fragment or analog thereof having the ability to bind to FRB or a fragment or analog thereof in the presence of rapamycin or an analog of rapamycin (e.g., RAD001) and having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or differ by NO more than 30, 25, 20, 15, 10, 5, 4, 3, 2 or 1 amino acid residue to the FKBP sequence of SEQ ID No. 148 or 149; the second switch domain comprises an FRB fragment or analog thereof having the ability to bind to FRB or a fragment or analog thereof in the presence of rapamycin or a rapamycin analog and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to or differ by NO more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue from the FRB sequence of SEQ ID NO: 150. In one embodiment, the RCAR described herein comprises a switch domain comprising the amino acid residues disclosed in SEQ ID NO:148 (or SEQ ID NO:149) and a switch domain comprising the amino acid residues disclosed in SEQ ID NO: 150.

In embodiments, the FKBP/FRB dimerization switch comprises a modified FRB switch domain that exhibits altered (e.g., enhanced) complex formation between an FRB-based switch domain (e.g., a modified FRB switch domain, an FKBP-based switch domain) and a dimerization molecule (e.g., rapamycin or a rapamycin analog, e.g., RAD 001). In one embodiment, the modified FRB switch domain comprises one or more mutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations selected from amino acid positions L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105 and F2108, wherein the wild-type amino acid is mutated to any other naturally occurring amino acid. In one embodiment, the mutant FRB comprises a mutation at E2032, wherein the E2032 mutation is to phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I) (e.g., SEQ ID NO:151) or leucine (E2032L) (e.g., SEQ ID NO: 152). In one embodiment, the mutant FRB comprises a mutation at T2098, wherein the T2098 mutation is to phenylalanine (T2098F) or leucine (T2098L), e.g., SEQ ID NO: 153. In one embodiment, the mutant FRB comprises mutations at E2032 and T2098, wherein E2032 is mutated to any amino acid and wherein T2098 is mutated to any amino acid, e.g., SEQ ID NO: 154. In one embodiment, the mutant FRB comprises the E2032I and T2098L mutations, e.g., SEQ ID NO: 155. In one embodiment, the mutant FRB comprises the E2032L and T2098L mutations, e.g., SEQ ID NO: 156.

Table 6 exemplary mutant FRBs with increased affinity for dimerized molecules.

Other suitable dimerization switches include gyrB-gyrB based dimerization switches, gibberellin based dimerization switches, tag/binder dimerization switches, and halo-tag/snap-tag dimerization switches. Such switches and related dimerization molecules will be apparent to one of ordinary skill in light of the guidance provided herein.

Dimerized molecules

The association between the switch domains is initiated by the dimerizing molecule. In the presence of the dimerization molecule, the interaction or association between the switch domains allows for signal transduction between a polypeptide associated (e.g., fused) to the first switch domain and a polypeptide associated (e.g., fused) to the second switch domain. In the presence of non-limiting levels of the dimerization molecule, signal transduction is increased by 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 5, 10, 50, 100 fold, e.g., as measured in a system as described herein.

Rapamycin and rapamycin analogs (sometimes referred to as rapa analogs), such as RAD001, can be used as a dimerizing molecule in the FKBP/FRB based dimerization switches described herein. In one embodiment the dimerising molecule may be selected from rapamycin (sirolimus), RAD001 (everolimus), zotarolimus (zotarolimus), temsirolimus, AP-23573 (ridolimus), temsirolimus (biolimus) and AP 21967. Additional rapamycin analogs suitable for use with FKBP/FRB-based dimerization switches are further described in the section entitled "combination therapy" or in the subsection entitled "use in combination with immunopotentiating low dose mTOR inhibitors".

Split CAR (split CAR)

In some embodiments, the CAR-expressing cell uses a dividing CAR. The split CAR protocol is described in more detail in publications WO 2014/055442 and WO 2014/055657, which are incorporated herein by reference. Briefly, the dividing CAR system comprises a cell that expresses a first CAR having a first antigen binding domain and a costimulatory domain (e.g., 41BB), and the cell further expresses a second CAR having a second antigen binding domain and an intracellular signaling domain (e.g., CD3 ζ). When the cell encounters the first antigen, the co-stimulatory domain is activated and the cell proliferates. When the cell encounters a second antigen, the intracellular signaling domain is activated and cell killing activity is turned on. Thus, CAR-expressing cells are fully activated only in the presence of both antigens. In embodiments, the first antigen binding domain recognizes CD33, e.g., comprises an antigen binding domain as described herein, and the second antigen binding domain recognizes an antigen expressed on acute myeloid leukemia cells, e.g., CD123, CLL-1, CD34, FLT3, or folate receptor beta. In embodiments, the first antigen binding domain recognizes CD33, e.g., comprises an antigen binding domain described herein, and the second antigen binding domain recognizes an antigen expressed on a B cell, e.g., CD19, CD20, CD22, or ROR 1.

Stability and mutation

The stability of a CD33 binding domain (e.g., an scFv molecule (e.g., a soluble scFv)) can be evaluated with reference to the biophysical properties (e.g., thermostability) of a conventional control scFv molecule or a full-length antibody. In one embodiment, the human scFv has a thermal stability in the assay that is about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees celsius, about 11 degrees celsius, about 12 degrees celsius, about 13 degrees celsius, about 14 degrees celsius, or about 15 degrees celsius higher than a control binding molecule (e.g., a conventional scFv molecule).

The improved thermostability of the CD33 binding domain (e.g., scFv) was subsequently conferred to the entire CAR33 construct, resulting in improved therapeutic properties of the CAR33 construct. The thermostability of the CD33 binding domain (e.g., scFv) can be improved by at least about 2 ℃ or 3 ℃ compared to conventional antibodies. In one embodiment, the CD33 binding domain (e.g., scFv) has improved thermostability at 1 ℃ compared to a conventional antibody. In another embodiment, the CD33 binding domain (e.g., scFv) has improved thermostability at 2 ℃ compared to a conventional antibody. In another embodiment, the scFv has improved thermostability at 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ℃ compared to a conventional antibody. Comparisons can be made, for example, between scFv molecules disclosed herein and full length antibodies. Thermal stability can be measured using methods known in the art. For example, in one embodiment, Tm may be measured. Methods for measuring Tm and other methods of determining protein stability are described in more detail below.

Mutations in the scFv alter the stability of the scFv and improve the overall stability of the scFv and CAR33 constructs. The stability of the humanized or human scFv was determined using measurements such as Tm, temperature denaturation and temperature aggregation.

The binding capacity of the mutant scFv can be determined using the assays described in the examples.

In one embodiment, the CD33 binding domain (e.g., scFv) comprises at least one mutation, such that the mutated scFv confers improved stability to the CAR33 construct. In another embodiment, the CD33 binding domain (e.g., scFv) comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations resulting from a humanization process, whereby the mutated scFv confers improved stability to the CAR33 construct.

Method for evaluating protein stability

The stability of the antigen binding domain can be assessed, for example, using the methods described below. Such methods allow for the determination of multiple thermal unfolding transitions, where the most unstable domain unfolds first or limits the overall stability threshold of a co-unfolded multidomain unit (e.g., a multidomain protein exhibiting a single unfolding transition). The most labile domain can be identified in a number of additional ways. Mutagenesis can be performed to detect which domain limits overall stability. In addition, protease resistance of multidomain proteins can be determined by means of DSC or other spectroscopic methods under conditions in which the most unstable domain is known to unfold intrinsically (Fontana et al, (1997) fold. Des.,2: R17-26; Dimasi et al, (2009) J.mol.biol.393: 672-692). Once the least stable domain is identified, a sequence encoding such a domain (or portion thereof) can be used as the test sequence in the method.

a) Thermal stability

The thermal stability of the composition can be analyzed using a number of non-limiting biophysical or biochemical techniques known in the art. In certain embodiments, thermal stability is evaluated by analytical spectroscopy.

One exemplary analytical spectroscopic method is Differential Scanning Calorimetry (DSC). DSC employs calorimeters that are sensitive to heat absorption that accompanies unfolding of most proteins or protein domains (see, e.g., Sanchez-Ruiz et al, Biochemistry,27:1648-52, 1988). To determine the thermostability of the protein, a sample of the protein was inserted into a calorimeter and the temperature was raised until the Fab or scFv unfolded. The temperature at which the protein unfolds is indicative of overall protein stability.

Another exemplary analytical spectroscopy method is Circular Dichroism (CD) spectroscopy. CD spectroscopy measures the optical activity of the composition as a function of increasing temperature. Circular Dichroism (CD) spectroscopy measures the difference in absorption of left-polarized light relative to right-polarized light due to structural asymmetry. Disordered or unfolded structures produce a CD spectrum that is very different from ordered or folded structures. The CD spectrum reflects the sensitivity of the protein to denaturing effects that increase temperature and thus indicates the thermal stability of the protein (see van Mierlo and Steemsma, J.Biotechnol.,79(3): 281-.

Another exemplary analytical spectroscopic method for measuring thermal stability is fluorescence emission spectroscopy (see van Mierlo and Steemsma, supra). Yet another exemplary analytical spectroscopic method for measuring thermal stability is Nuclear Magnetic Resonance (NMR) spectroscopy (see, e.g., van Mierlo and Steemsma, supra).

The thermal stability of the composition can be measured biochemically. An exemplary biochemical method for assessing thermal stability is a thermal challenge assay. In the "heat attack assay", the composition is subjected to a series of elevated temperatures for a set period of time. For example, in one embodiment, the subject scFv molecule or molecule comprising an scFv molecule is subjected to a series of increased temperatures for, e.g., 1-1.5 hours. The activity of the protein is then analyzed by relevant biochemical assays. For example, if the protein is a binding protein (e.g., an scFv or polypeptide comprising an scFv), the binding activity of the binding protein can be determined by functional or quantitative ELISA.

Such assays can be performed in a high throughput format and those using E.coli and high throughput screening are disclosed in the examples. Libraries of CD33 binding domains (e.g., scFv variants) can be generated using methods known in the art. CD33 binding domain (e.g., scFv) expression can be induced and CD33 binding domain (e.g., scFv) can be heat-challenged. Binding may be assayed for challenge in test samples and those stable CD33 binding domains (e.g., scFv) may be amplified and further characterized.

Thermal stability is assessed by measuring the melting temperature (Tm) of the composition using any of the techniques previously described (e.g., analytical spectroscopy techniques). The melting temperature is the temperature at the midpoint of the thermal transition curve at which 50% of the molecules of the composition are in the folded state (see, e.g., Dimasi et al, (2009) J.mol biol.393: 672-. In one embodiment, the Tm value of a CD33 binding domain (e.g., scFv) is about 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃, 91 ℃, 92 ℃, 93 ℃, 94 ℃, 95 ℃, 96 ℃, 97 ℃, 98 ℃, 100 ℃. In one embodiment, the Tm value of an IgG is about 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃, 91 ℃, 92 ℃, 93 ℃, 94 ℃, 95 ℃, 96 ℃, 97 ℃, 98 ℃, 99 ℃, 100 ℃. In one embodiment, the Tm value of the multivalent antibody is about 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃, 91 ℃, 92 ℃, 93 ℃, 94 ℃, 95 ℃, 96 ℃, 97 ℃, 98 ℃, 99 ℃, 100 ℃.

Thermal stability is also evaluated by measuring the specific heat or heat capacity (Cp) of the composition using analytical calorimetric techniques, such as DSC. The specific heat of the composition is the energy (e.g., in kcal/mol) required to raise the temperature of 1 mole of water by 1 ℃. Large Cp is an indicator of denatured or inactive protein composition. The change in heat capacity (Δ Cp) of the composition is measured by determining the specific heat of the composition before and after its thermal transition. Thermal stability can also be assessed by measuring or determining other parameters of thermodynamic stability, including unfolding Gibbs free energy (Δ G), unfolding enthalpy (Δ H), or unfolding entropy (Δ S). One or more of the above biochemical assays (e.g., a thermal challenge assay) are used to determine that 50% of the composition retains its activity(e.g., binding activity) temperature (i.e., T_CValue).

Furthermore, mutation to the CD33 binding domain (e.g., scFv) alters the thermostability of the CD33 binding domain (e.g., scFv) compared to the unmutated CD33 binding domain (e.g., scFv). When a humanized or human CD33 binding domain (e.g., scFv) is incorporated into the CAR33 construct, the CD33 binding domain (e.g., humanized or human scFv) confers thermal stability to the entire CD33 CAR construct. In one embodiment, the CD33 binding domain (e.g., scFv) comprises a single mutation that confers thermostability to the CD33 binding domain (e.g., scFv). In another embodiment, the CD33 binding domain (e.g., scFv) comprises multiple mutations that confer thermostability to the CD33 binding domain (e.g., scFv). In one embodiment, multiple mutations in a CD33 binding domain (e.g., scFv) have an additive effect on the thermostability of the CD33 binding domain (e.g., scFv).

b) Aggregate%

The stability of the composition can be determined by measuring its tendency to aggregate. Aggregation can be measured by a number of non-limiting biochemical or biophysical techniques. For example, the composition can be evaluated for aggregation using chromatography, such as Size Exclusion Chromatography (SEC). SEC separates molecules based on size. The column is packed with semi-solid beads of polymeric gel that allow ions and small molecules to enter the interior, but not large entities. When the protein composition is applied to the top of the column, the compact folded protein (i.e., unaggregated protein) is distributed throughout the larger volume of solvent available for large aggregates of protein. Thus, large aggregates move more rapidly through the column, and in this way the mixture can be separated or fractionated into its components. Each fraction can be quantified separately (e.g., by light scattering) as it elutes from the gel. Thus, the% aggregation of the composition can be determined by comparing the concentration of the fractions with the total concentration of protein applied to the gel. The stable composition elutes from the column as a substantially single fraction and appears as a substantially single peak in the elution profile or chromatogram.

c) Binding affinity

The stability of the composition can be assessed by determining its target binding affinity. A wide variety of methods for determining binding affinity are known in the art. One exemplary method for determining binding affinity employs surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows analysis of real-time biospecific interactions by detecting changes in protein concentration inside the Biosensor matrix (e.g., using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, n.j.)). For further description, see Jonsson, U.S. et al, (1993) Ann.biol.Clin.51: 19-26; jonsson, U.S., i (1991) Biotechniques 11: 620-; johnson, B.et al, (1995) J.mol.Recognit.8: 125-.

In one aspect, the antigen binding domain of the CAR comprises an amino acid sequence that is homologous to an antigen binding domain amino acid sequence described herein, and the antigen binding domain retains the desired functional properties of a CD33 antibody fragment described herein. In a particular aspect, the CAR composition of the invention comprises an antibody fragment. In yet another aspect, the antibody fragment comprises an scFv.

In various aspects, the antigen binding domain of a CAR can be engineered by modifying one or more amino acids within one or both variable regions (e.g., VH and/or VL), e.g., within one or more CDR regions and/or within one or more framework regions. In a particular aspect, the CAR composition of the invention comprises an antibody fragment. In yet another aspect, the antibody fragment comprises an scFv.

One of ordinary skill in the art will appreciate that the antibodies or antibody fragments of the invention may also be modified such that they vary in amino acid sequence (e.g., from wild-type), but do not vary in the desired activity. For example, the protein may be subjected to additional nucleotide substitutions that result in amino acid substitutions at "non-essential" amino acid residues. For example, a non-essential amino acid residue in a molecule can be replaced with another amino acid residue from the same side chain family. In another embodiment, an amino acid fragment can be replaced with a structurally similar fragment that differs in the order and composition of the side chain family members, e.g., conservative substitutions can be made in which amino acid residues are replaced with amino acid residues having similar side chains.

Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).

In the context of two or more nucleic acid or polypeptide sequences, a percent identity refers to two or more sequences that are the same. Two sequences are "substantially identical" if they have a specified percentage of identical amino acid residues or nucleotides (e.g., 60% identity, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over the specified region or over the entire sequence when not specified) as measured for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used or alternative parameters may be specified. Based on the program parameters, the sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence. Methods for aligning sequences for comparison are well known in the art. Optimal sequence alignments for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, (1970) adv.Appl.Math.2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J.mol.biol.48:443, by Pearson and Lipman, (1988) similarity search method of Proc.nat' l.Acad.Sci.USA 85:2444, by computerized execution of these algorithms (Wisconsin Genetics software package, Genetics Computer Group,575 Science Dr., Madison, GAP in WI, BESTFIT, FASTA and TFASTA) or by manual alignment and visual inspection (see, for example, Brent et al 2003, (Current Protocols in Molecular Biology).

Two examples of algorithms suitable for determining sequence identity and percent sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, respectively, in Altschul et al, (1977) Nuc. acids Res.25: 3389-; and Altschul et al, (1990) J.mol.biol.215: 403-. Software for performing BLAST analyses is publicly available through the national center for biotechnology information.

The percent identity between two amino acid sequences can also be determined using the e.meyers and w.miller algorithms (1988) comp.appl.biosci.4: 11-17) that have been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, gap length penalty of 12, and gap penalty of 4). Furthermore, the percent identity between two amino acid sequences can be determined using the Needlema and Wunsch (1970) J.mol.biol.48: 444-.

In one aspect, the invention contemplates amino acid sequence modifications of the starting antibody or fragment (e.g., scFv) that result in a functionally equivalent molecule. For example, the VH or VL of the CD33 binding domain (e.g., scFv) included in the CAR can be modified to retain at least about 70%, 71% of the original VH or VL framework region as compared to the CD33 binding domain (e.g., scFv). 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. The invention contemplates modifying the entire CAR construct, e.g., in one or more amino acid sequences of multiple domains of the CAR construct, with the intent of producing a functionally equivalent molecule. The CAR construct can be modified to retain at least about 70%, 71% of the starting CAR construct. 72 percent. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

RNA transfection

Disclosed herein are methods for generating in vitro transcribed RNA CARs. The invention also includes an RNA construct encoding a CAR that can be transfected directly into a cell. One method of generating mRNA for transfection may involve In Vitro Transcription (IVT) of the template with specially designed primers, followed by addition of poly A, to generate constructs containing 3 ' and 5 ' untranslated sequences ("UTR"), a 5 ' cap and/or an Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a poly A tail, typically 50-2000 bases in length (SEQ ID NO: 35). The RNA thus produced can efficiently transfect cells of various kinds. In one aspect, the template comprises a sequence for a CAR.

In one aspect, the CD33 CAR is encoded by messenger rna (mrna). In one aspect, mRNA encoding a CD33 CAR is introduced into an immune effector cell (e.g., a T cell or NK cell) to generate a CAR-expressing cell (e.g., a CART cell or a CAR-expressing NK cell).

In one embodiment, the in vitro transcribed RNA CAR can be introduced into the cell as a form of transient transfection. RNA is produced by in vitro transcription using a Polymerase Chain Reaction (PCR) generated template. DNA of interest from any source can be directly converted to template by PCR to synthesize mRNA in vitro using appropriate primers and RNA polymerase. The source of DNA may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable source of DNA. The template of interest for in vitro transcription is the CAR of the invention. For example, a template for an RNA CAR comprises an extracellular region comprising a single-stranded variable domain of an anti-tumor antibody; a hinge region, a transmembrane domain (e.g., the transmembrane domain of CD8 a); and a cytoplasmic region comprising an intracellular signaling domain (e.g., comprising the signaling domain of CD 3-zeta and the signaling domain of 4-1 BB).

In one embodiment, the DNA to be used in PCR contains an open reading frame. The DNA may be derived from a naturally occurring DNA sequence derived from the genome of an organism. In one embodiment, the nucleic acid may include some or all of the 5 'untranslated region and/or the 3' untranslated region (UTR). Nucleic acids may include exons and introns. In one embodiment, the DNA to be used for PCR is a human nucleic acid sequence. In another embodiment, the DNA to be used in PCR is a human nucleic acid sequence, including a 5'UTR and a 3' UTR. The DNA may alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is an artificial DNA sequence comprising gene portions that are joined together to form an open reading frame encoding a fusion protein. The individual DNA portions that are linked together may be from a single organism or from more than one organism.

PCR is used to generate templates for in vitro transcription of mRNA for transfection. Methods for performing PCR are well known in the art. The primers used in PCR are designed to have a region that is substantially complementary to a region of DNA to be used as a template for PCR. As used herein, "substantially complementary" refers to a nucleotide sequence in which most or all of the bases in the primer sequence are complementary or one or more bases are non-complementary or mismatched. The substantially complementary sequence is capable of renaturing or hybridizing to the intended DNA target under the renaturation conditions used in PCR. The primer can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify the portion of nucleic acid (open reading frame) normally transcribed in a cell, including the 5 'and 3' UTRs. Primers can also be designed to amplify a portion of the nucleic acid encoding a particular domain of interest. In one embodiment, the primers are designed to amplify coding regions of human cDNA, including all or part of the 5 'and 3' UTRs. Primers for PCR can be generated by synthetic methods well known in the art. A "forward primer" is a primer that contains a region of nucleotides that is substantially complementary to a nucleotide upstream of a DNA sequence to be amplified on a DNA template. "upstream" is used herein to refer to a position 5' to the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that contains a region of nucleotides that is substantially complementary to a double-stranded DNA template downstream of the DNA sequence to be amplified on the DNA template. "downstream" is used herein to refer to a position 3' to the DNA sequence to be amplified relative to the coding strand.

Any DNA polymerase used for PCR can be used in the methods disclosed herein. Reagents and polymerases are commercially available from a number of sources.

Chemical structures having the ability to promote stability and/or translation efficiency may also be used. The RNA preferably has a 5'UTR and a 3' UTR. In one embodiment, the 5' UTR is between 1 and 3000 nucleotides in length. The length of the 5'UTR and 3' UTR sequences to be added to the coding region can be varied by different methods including, but not limited to, designing primers for PCR that anneal to different regions of the UTR. Using this method, one of ordinary skill in the art can adjust the length of the 5'UTR and 3' UTR required to achieve optimal translational efficiency after transfection of transcribed RNA.

The 5'UTR and 3' UTR may be naturally occurring, endogenous 5'UTR and 3' UTR of the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest may be added by incorporating the UTR sequences into the forward and reverse primers or by any other modification to the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest can be used to modulate the stability and/or translational efficiency of the RNA. For example, AU-rich elements in the 3' UTR sequence are known to reduce mRNA stability. Thus, based on UTR properties well known in the art, the 3' UTR may be selected or designed to increase the stability of the transcribed RNA.

In one embodiment, the 5' UTR may contain a Kozak sequence of an endogenous nucleic acid. Alternatively, when a 5'UTR that is not endogenous to the nucleic acid of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding a 5' UTR sequence. Kozak sequences may increase the translation efficiency of certain RNA transcripts, but do not appear to be required for efficient translation of all RNAs. It is known in the art that Kozak sequences are required for many mrnas. In other embodiments, the 5'UTR may be a 5' UTR of an RNA virus whose RNA genome is stable in the cell. In other embodiments, a variety of nucleotide analogs can be used in the 3 'or 5' UTRs to hinder exonuclease degradation of mRNA.

In order to be able to synthesize RNA from a DNA template without the need for gene cloning, a transcription promoter should be linked to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as an RNA polymerase promoter is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In a preferred embodiment, the promoter is the T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, the T3RNA polymerase promoter and the SP6RNA polymerase promoter. Consensus nucleotide sequences for the T7, T3, and SP6 promoters are known in the art.

In a preferred embodiment, the mRNA has caps at both the 5 'end and the 3' poly A tail that determine ribosome binding, translation initiation, and mRNA stability in the cell. On circular DNA templates, e.g., plasmid DNA, RNA polymerase produces long concatemer products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3' UTR produces mRNA of a size not normally effective in eukaryotic transfection, even if it is polyadenylated after transcription.

On a linear DNA template, phage T7RNA polymerase can extend the 3' end of the transcript past the last base of the template (Schenborn and Mieredorf, Nuc Acids Res.,13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. biochem.,270:1485-65 (2003)).

A common method for integrating a polyA/T fragment into a DNA template is molecular cloning. However, the polyA/T sequences incorporated into plasmid DNA may cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells often suffer from high contamination by deletions and other aberrations. This makes cloning methods not only cumbersome and time consuming, but often unreliable. This is why the following approach is urgently needed: allows the construction of DNA templates with polyA/T3' fragments without highly desirable cloning.

PolyA/T segments of DNA templates for transcription can be generated during PCR by using reverse primers containing a poly-thymidylate tail (e.g., 100T tail (SEQ ID NO:31) (which can be 50-5000T in size (SEQ ID NO:32)) or by any other method after PCR including, but not limited to, DNA ligation or in vitro recombination.

The poly A tail of RNA can be further extended after in vitro transcription using a poly A polymerase such as E.coli poly A polymerase (E-PAP). In one embodiment, increasing the length of the poly A tail from 100 nucleotides to between 300 and 400 nucleotides (SEQ ID NO:34) results in about a two-fold increase in the translation efficiency of the RNA. In addition, conjugation of different chemical groups to the 3' end can increase mRNA stability. Such conjugation may contain modified/artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into a polya tail using polya polymerase. ATP analogs may also increase the stability of RNA.

The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein comprises a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot et al, Trends in biochem. Sci.,29:436- & 444 (2001); Stepinski et al, RNA,7:1468-95 (2001); Elango et al, Biochim. Biophys. Res. Commun.,330:958- & 966 (2005)).

The RNA produced by the methods disclosed herein may also contain an Internal Ribosome Entry Site (IRES) sequence. The IRES sequence can be any viral sequence, chromosomal sequence, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and facilitates initiation of translation. Any solute suitable for electroporation of cells may be included, which may contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

RNA can be introduced into the target cell using any of a number of different methods, such as, but not limited to, commercially available methods including: electroporation (Amaxa Nuclear of choice-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830(BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection or biolistic particle delivery systems such as "Gene guns" (see, e.g., Nishikawa et al, Hum Gene Ther, 12(8):861-70 (2001)).

Non-viral delivery method

In some aspects, a non-viral approach can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or subject.

In some embodiments, the non-viral method comprises the use of a transposon (also referred to as a transposable element). In some embodiments, a transposon is a piece of DNA that can insert itself into a location in the genome, e.g., a piece of DNA that can self-replicate and insert its copy into the genome or a piece of DNA that can be excised from a longer nucleic acid and inserted into another location in the genome. For example, transposons comprise DNA sequences consisting of inverted repeats which flank the gene for transposition.

Exemplary methods of nucleic acid delivery using transposons include the Sleeping Beauty Transposon System (SBTS) and piggybac (pb) transposon system. See, e.g., Aronovich et al, hum.mol.genet.20.R1(2011): R14-20; singh et al, Cancer Res.15(2008): 2961-; huang et al, mol. ther.16(2008): 580-; grabundzija et al, mol. ther.18(2010): 1200-1209; kebriaiei et al, blood.122.21(2013): 166; molecular Therapy 16.9(2008): 1515-16; bell et al, nat. Protoc.2.12(2007): 3153-65; and Ding et al, cell.122.3(2005):473-83, all of which are incorporated herein by reference.

The SBTS comprises two components: 1) a transposon containing the transgene and 2) a source of transposase. Transposases can transpose transposons from a vector plasmid (or other donor DNA) to a target DNA, such as a host cell chromosome/genome. For example, the transposase is combined with the vector plasmid/donor DNA, the transposon (containing the transgene)) is excised from the plasmid and inserted into the genome of the host cell. See, e.g., Aronovich et al, supra.

Exemplary transposons include pT 2-based transposons. See, e.g., Grabundzija et al, Nucleic Acids Res.41.3(2013): 1829-47; and Singh et al, Cancer Res.68.8(2008): 2961-. Exemplary transposases include Tc 1/water hand transposase, e.g., SB10 transposase or SB11 transposase (an overactive transposase that can be expressed, for example, from a cytomegalovirus promoter). See, e.g., Aronovich et al; kebriaiei et al; and Grabundzija et al, which are all incorporated herein by reference.

The use of SBTS allows for efficient integration and expression of transgenes, e.g., nucleic acids encoding the CARs described herein. Provided herein are methods of generating cells (e.g., T cells or NK cells) that stably express a CAR described herein, e.g., using a transposon system such as SBTS.

According to the methods described herein, in some embodiments, one or more nucleic acids (e.g., plasmids) containing an SBTS component are delivered to a cell (e.g., a T cell or NK cell). For example, the nucleic acid is delivered by standard nucleic acid (e.g., plasmid DNA) delivery methods (e.g., the methods described herein, e.g., electroporation, transfection, or lipofection). In some embodiments, the nucleic acid contains a transposon that comprises a transgene (e.g., a nucleic acid encoding a CAR described herein). In some embodiments, the nucleic acid contains a transposon that comprises a transgene (e.g., a nucleic acid encoding a CAR described herein) and a nucleic acid sequence encoding a transposase. In other embodiments, a system is provided having two nucleic acids (e.g., a two plasmid system), e.g., where a first plasmid contains a transposon that comprises the transgene and a second plasmid contains a nucleic acid sequence encoding the transposase. For example, the first nucleic acid and the second nucleic acid are co-delivered into the host cell.

In some embodiments, cells (e.g., T cells or NK cells) expressing the CARs described herein are generated by using a combination of gene insertion methods employing SBTS and genetic editing methods employing nucleases (e.g., Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas systems, or engineered meganucleases (meganucleases), re-engineered homing endonucleases).

In some embodiments, the use of non-viral delivery methods allows reprogramming of cells (e.g., T cells or NK cells) and direct infusion of cells into a subject. Advantages of non-viral vectors include, but are not limited to, convenience and relatively low cost in producing sufficient quantities required to meet patient population, stability during storage, and lack of immunogenicity.

Nucleic acid constructs encoding CAR

The invention also provides nucleic acid molecules encoding one or more of the CAR constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct.

Thus, in one aspect, the invention relates to an isolated nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises a CD33 binding domain (e.g., a humanized or human CD33 binding domain), a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain (e.g., a costimulatory signaling domain) and/or a primary signaling domain (e.g., a zeta chain). In one embodiment, the CD33 binding domain is a CD33 binding domain described herein, e.g., a CD33 binding domain comprising a sequence selected from SEQ ID NOs 39-47 or a sequence 95-99% identical thereto. In one embodiment, the transmembrane domain is a transmembrane domain of a protein, e.g., as described herein, e.g., selected from the group consisting of the α, β, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD 154. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO 6 or a sequence 95-99% identical thereto. In one embodiment, the CD33 binding domain is connected to the transmembrane domain by a hinge region (e.g., a hinge region as described herein). In one embodiment, the hinge region comprises

SEQ ID NO

2 or 3 or 4 or 5 or a sequence with 95-99% identity thereto. In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a co-stimulatory domain. In one embodiment, the co-stimulatory domain is a functional signaling domain of a protein, e.g., described herein, e.g., selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signal molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1(CD11a/CD a), 4-1BB (CD137), B a-H a, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHT TR), KIRDS a, SLAMF a, NKp a (KLRF a), NKp a, CD a alpha, CD a beta, IL2 a gamma, ITIL 7 alpha, IT3672, VLITGA 72, VLITGA a, GAITGB 72, GAITGA a, GAITGB 11-a, GAITGA a, GAITGB 11, GAITGA a, GAITGB a, GAIT11, CD a, GAITGA a, GAITGB 72, GAITGA a, CD a, GAITGB 11-a, GAITGB 11, GAITGA a, GAITGB a, CD a, GAITGA a, GAITGB 11-a, GAITCD a, GAITGA a, CD a, GAITGB 11-a, CD a, GAITGB 11, SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds to CD 83. In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO. 7 or a sequence 95-99% identical thereto. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of CD3 ζ. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO 7 or SEQ ID NO 8 or a sequence 95-99% identical thereto and the sequence of SEQ ID NO 9 or SEQ ID NO 10 or a sequence 95-99% identical thereto, wherein the sequences comprising the intracellular signaling domain are expressed in frame and as a single polypeptide chain.

In another aspect, the invention relates to an isolated nucleic acid molecule encoding a CAR construct comprising a leader sequence of SEQ ID NO:1, a hinge region having a sequence selected from SEQ ID NO:39-47 (or a sequence 95-99% identical thereto), SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 (or a sequence 95-99% identical thereto), a CD27 co-stimulatory domain having a sequence of SEQ ID NO:6 (or a sequence 95-99% identical thereto), a 4-1BB co-stimulatory domain having a sequence of SEQ ID NO:7 or a transmembrane domain having a sequence of SEQ ID NO:8 (or a sequence 95-99% identical thereto) or a CD28 co-stimulatory domain having a sequence of SEQ ID NO:379 (or a sequence 95-99% identical thereto), an isolated nucleic acid molecule encoding a CAR construct comprising a leader sequence of SEQ ID NO:1, a hinge region of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:5 (or a sequence 95-99% identical thereto) Or an ICOS co-stimulatory domain having the sequence of SEQ ID NO:381 (or a sequence with 95-99% identity thereto), and a CD3 zeta stimulatory domain having the sequence of SEQ ID NO:9 or SEQ ID NO:10 (or a sequence with 95-99% identity thereto).

In another aspect, the invention relates to an isolated polypeptide molecule encoded by the nucleic acid molecule. In one embodiment, the isolated polypeptide molecule comprises a sequence selected from SEQ ID NOS 48-56 or a sequence 95-99% identical thereto.

In another aspect, the invention relates to a nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR) molecule comprising a CD33 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain, and wherein the CD33 binding domain comprises a sequence selected from SEQ ID NOs 75-83 or a sequence 95-99% identical thereto. In one embodiment, the encoded CAR molecule further comprises a sequence encoding a co-stimulatory domain. In one embodiment, the co-stimulatory domain is a functional signaling domain of a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signal molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1(CD11a/CD a), 4-1BB (CD137), B a-H a, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHT TR), KIRDS a, SLAMF a, NKp a (KLRF a), NKp a, CD a alpha, CD a beta, IL2 a gamma, ITIL 7 alpha, IT3672, VLITGA 72, VLITGA a, GAITGB 72, GAITGA a, GAITGB 11-a, GAITGA a, GAITGB 11, GAITGA a, GAITGB a, GAIT11, CD a, GAITGA a, GAITGB 72, GAITGA a, CD a, GAITGB 11-a, GAITGB 11, GAITGA a, GAITGB a, CD a, GAITGA a, GAITGB 11-a, GAITCD a, GAITGA a, CD a, GAITGB 11-a, CD a, GAITGB 11, SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds to CD 83. In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO 7. In one embodiment, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of a T cell receptor, CD epsilon, CD134, CD137, CD154, KIR2DS, OX, CD, LFA-1(CD and CD), ICOS (CD278), 4-1BB (CD137), GITR, CD, BAFFR, HVEM (LITR), SLAMF, NKp (KLRF), NKp, CD160, CD, IL2 β, IL2 (common γ), IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, LFA-1, GAM, GAMMA, CD11, ACAGX, ACAGG, CD229, ITGA, VLA-6, CD49, ITGAGB, ITGAMMA, CD11, ITGABYBYBYGAE, CD103, ITGAB, CD11, ITGAMMA, CD-1, CD-CD, ITGAGB, ITGAX, CD-CD (CD-CD, PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, and PAG/Cbp.

In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO 6. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of zeta. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO. 7 and the sequence of SEQ ID NO. 9, wherein the sequences comprising the intracellular signaling domain are expressed in frame and as a single polypeptide chain. In one embodiment, the CD33 binding domain is connected to the transmembrane domain by a hinge region. In one embodiment, the hinge region comprises SEQ ID NO 2. In one embodiment, the hinge region comprises SEQ ID NO 3 or SEQ ID NO 4 or SEQ ID NO 5.

In another aspect, the invention relates to an encoded CAR molecule comprising the leader sequence of SEQ ID NO. 1, a scFv domain having a sequence selected from SEQ ID NO. 39-47 or a sequence with 95-99% identity thereto, the hinge region of SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 5, a transmembrane domain having the sequence of SEQ ID NO. 6, a 4-1BB costimulatory domain having the sequence of SEQ ID NO. 7 or a CD27 costimulatory domain having the sequence of SEQ ID NO. 8, and a CD3 zeta stimulatory domain having the sequence of SEQ ID NO. 9 or SEQ ID NO. 10. In one embodiment, the encoded CAR molecule comprises a sequence selected from SEQ ID NOS 48-56 or a sequence 95-99% identical thereto.

Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by deriving the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically, rather than clonally.

The present invention also provides a vector into which the DNA of the present invention is inserted. Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of the transgene and its propagation in progeny cells. Lentiviral vectors have the added advantage over vectors derived from cancer-retroviruses (e.g., murine leukemia virus) in that they can transduce non-proliferative cells, such as hepatocytes. They also have the added advantage of low immunogenicity. Retroviral vectors may also be, for example, gamma retroviral vectors. The gamma retroviral vector may, for example, comprise a promoter, a packaging signal (ψ), a Primer Binding Site (PBS), one or more (e.g., two) Long Terminal Repeats (LTRs), and a transgene of interest, e.g., a gene encoding a CAR. The gamma retroviral vector may lack viral structural genes such as gag, pol, and env. Exemplary gamma retroviral vectors include Murine Leukemia Virus (MLV), Spleen Focus Forming Virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. Other gamma retroviral Vectors are described, for example, in Tobias Maetzig et al, "Gamma aretroviral Vectors: Biology, Technology and Application" Virus.2011 Jun; 677 and 713 (3), (6).

In another embodiment, the vector comprising a nucleic acid encoding a CAR desired in the present invention is an adenoviral vector (A5/35). In another embodiment, expression of the nucleic acid encoding the CAR can be accomplished using transposons such as sleeping beauty, cripper, CAS9, and zinc finger nucleases. See June et al, 2009Nature Reviews Immunology 9.10:704-716, below, which is incorporated herein by reference.

Briefly, expression of a natural or synthetic nucleic acid encoding a CAR is generally achieved by operably linking a nucleic acid encoding the CAR polypeptide or portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be adapted for replication and integration in eukaryotes. Common cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequence.

The expression constructs of the invention can also be used for nucleic acid immunisation and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector.

Nucleic acids can be cloned into numerous types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

In addition, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al, 2012, Molecular CLONING: A Laboratory Manual, Vol.1-4, Cold Spring Harbor Press, NY) and in other virology and MOLECULAR biology MANUALs. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of a subject in vivo or ex vivo. Numerous retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Numerous adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Typically, these are located in the region 30-110bp upstream of the start site, although numerous promoters have been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, such that promoter function is preserved when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter, the individual elements appear to act synergistically or independently to activate transcription. Exemplary promoters include the CMV IE gene promoter, the EF-1 alpha promoter, the ubiquitin C promoter, or the phosphoglycerate kinase (PGK) promoter.

An example of a promoter capable of expressing a CAR transgene in mammalian T cells is the EF1a promoter. The native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNA to the ribosome. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to efficiently drive expression of CARs from transgenes cloned into lentiviral vectors. See, e.g., Milone et al, mol. ther.17(8): 1453-. In one aspect, the EF1a promoter comprises the sequence provided as SEQ ID NO 11.

Another example of a promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. This promoter sequence is a constitutively strong promoter sequence capable of driving high levels of expression of any polynucleotide sequence to which it is operably linked. However, other constitutive promoter sequences may also be used, including, but not limited to, monkey virus 40(SV40) early promoter, Mouse Mammary Tumor Virus (MMTV), Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, elongation factor-1 α promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of the polynucleotide sequence to which it is operably linked when such expression is desired or turning off expression when such expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

Another example of a promoter is the phosphoglycerate kinase (PGK) promoter. In embodiments, a truncated PGK promoter (e.g., a PGK promoter having a deletion of one or more, e.g., 1, 2, 5, 10, 100, 200, 300, or 400 nucleotides when compared to a wild-type PGK promoter sequence) may be desired. The nucleotide sequences of exemplary PGK promoters are provided below.

WT PGK promoter

ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCCCATCGCCGCTTCTAGGCCCACTGCGACGCTTGCCTGCACTTCTTACACGCTCTGGGTCCCAGCCGCGGCGACGCAAAGGGCCTTGGTGCGGGTCTCGTCGGCGCAGGGACGCGTTTGGGTCCCGACGGAACCTTTTCCGCGTTGGGGTTGGGGCACCATAAGCT

(SEQ ID NO:384)

Exemplary truncated PGK promoters：

PGK100:ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTG

(SEQ ID NO:385)

PGK200:

ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACG

(SEQ ID NO:386)

PGK300:

ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCG

(SEQ ID NO:387)

PGK400:

ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCCCATCGCCGCTTCTAGGCCCACTGCGACGCTTGCCTGCACTTCTTACACGCTCTGGGTCCCAGCCG

(SEQ ID NO:388)

The vector may also include, for example, signal sequences to facilitate secretion, polyadenylation signals, and transcription terminators (e.g., from the Bovine Growth Hormone (BGH) gene), elements that permit episomal replication and replication in prokaryotes (e.g., the SV40 origin of replication and ColE1 or other elements known in the art), and/or elements that permit selection (e.g., the ampicillin resistance gene and/or zeocin marker).

To assess the expression of the CAR polypeptide or portion thereof, the expression vector to be introduced into the cells can also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of cells exhibiting expression from a population of cells sought to be transfected or infected with the viral vector. In other aspects, the selectable marker may be carried on a separate DNA fragment and used in a co-transfection procedure. The selectable marker and reporter gene may be flanked by appropriate regulatory sequences capable of effecting expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue and that encodes a polypeptide whose expression is displayed by some readily detectable property (e.g., enzymatic activity). Expression of the reporter gene is determined at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al, 2000FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Typically, the construct with the least 5' flanking region that showed the highest level of reporter expression is identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of various substances to modulate promoter-driven transcription.

In one embodiment, the vector may further comprise a nucleic acid encoding a second CAR. In one embodiment, the second CAR comprises an antigen binding domain against a target expressed on acute myeloid leukemia cells (e.g., CD123, CD34, CLL-1, FLT3, or folate receptor beta). In one embodiment, the vector comprises a nucleic acid sequence encoding a first CAR that specifically binds a first antigen and includes an intracellular signaling domain with a costimulatory signaling domain but no primary signaling domain, and a nucleic acid sequence encoding a second CAR that specifically binds a second, different antigen and includes an intracellular signaling domain with a primary signaling domain but no costimulatory signaling domain. In one embodiment, the vector comprises a nucleic acid encoding a first CD33 CAR comprising a CD33 binding domain, a transmembrane domain, and a costimulatory domain, and a nucleic acid encoding a second CAR that specifically binds to an antigen other than CD33 (e.g., an antigen expressed on AML cells, e.g., CD123, CD34, CLL-1, FLT3, or folate receptor beta) and comprises an antigen binding domain, a transmembrane domain, and a primary signal domain. In another embodiment, the vector comprises a nucleic acid encoding a first CD33 CAR comprising a CD33 binding domain, a transmembrane domain, and a primary signal domain, and a nucleic acid encoding a second CAR that specifically binds to an antigen other than CD33 (e.g., an antigen expressed on AML cells, e.g., CD123, CD34, CLL-1, FLT3, or folate receptor beta) and comprises an antigen binding domain, a transmembrane domain, and a costimulatory signal domain for the antigen.

In one embodiment, the vector comprises a nucleic acid encoding a CD33 CAR described herein and a nucleic acid encoding an inhibitory CAR. In one embodiment, the inhibitory CAR comprises an antigen binding domain that binds to an antigen that is present on normal cells, but not on cancer cells, e.g., normal cells that also express CD 33. In one embodiment, the inhibitory CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. For example, the intracellular domain of an inhibitory CAR may be an intracellular domain of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR β.

In embodiments, the vector may comprise two or more nucleic acid sequences encoding one CAR, e.g., a CD33 CAR described herein, and a second CAR, e.g., an inhibitory CAR or a CAR that specifically binds to an antigen other than CD33 (e.g., an antigen expressed on AML cells, e.g., CD123, CLL-1, CD34, FLT3, or folate receptor beta). In such embodiments, the two or more nucleic acid sequences encoding the CAR are encoded by a single nucleic acid molecule in the same open reading frame and as a single polypeptide chain. In this aspect, two or more CARs, for example, can be separated by one or more peptide cleavage sites (e.g., self cleavage sites or substrates of intracellular proteases). Examples of peptide cleavage sites include the following sequences, wherein GSG residues are optional:

T2A:(GSG)E G R G S L L T C G D V E E N P G P(SEQ ID NO:389)

P2A:(GSG)A T N F S L L K Q A G D V E E N P G P(SEQ ID NO:390)

E2A:(GSG)Q C T N Y A L L K L A G D V E S N P G P(SEQ ID NO:391)

F2A:(GSG)V K Q T L N F D L L K L A G D V E S N P G P(SEQ ID NO:392)

Methods for introducing and expressing genes into cells are known in the art. In the case of expression vectors, the vectors can be readily introduced into host cells (e.g., mammalian cells, bacterial cells, yeast cells, or insect cells) by any method known in the art. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al, 2012, Molecular CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for introducing the polynucleotide into the host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA vectors and RNA vectors. Viral vectors and particularly retroviral vectors have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles). Other methods of targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable submicrometer-scale delivery systems.

Where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo or in vivo) is contemplated. In another aspect, the nucleic acid can be bound to a lipid. The nucleic acid associated with a lipid may be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, linked to a liposome via a linker molecule that binds both the liposome and the oligonucleotide, embedded within a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in a micelle, or complexed with a micelle or associated with a lipid. The composition associated with the lipid, lipid/DNA or lipid/expression vector is not limited to any particular structure in solution. For example, they may exist as bilayer structures, as micelles, or in "collapsed" structures. They may also simply be dispersed in the solution b, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which may be naturally occurring lipids or synthetic lipids. For example, lipids include fatty droplets that naturally occur in the cytoplasm and compounds that contain long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") is available from Sigma, st.louis, MO; dicetyl phosphate ("DCP") is available from K & K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other Lipids are available from Avanti Polar Lipids, Inc. The mother liquor of lipids in chloroform or chloroform/methanol may be stored at about-20 ℃. Chloroform was used as the only solvent because it evaporates more readily than methanol. "liposomes" is a generic term that encompasses a variety of single and multilamellar lipid carriers formed by the creation of closed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous media. When phospholipids are suspended in excess aqueous solution, they form spontaneously. Lipid components rearrange themselves before forming closed structures and entrap water and dissolved solutes between lipid bilayers (Ghosh et al, 1991 Glycobiology 5: 505-10). However, compositions having a structure in solution that is different from the structure of normal vesicles are also contemplated. For example, lipids may adopt a micellar structure or simply exist as non-uniform aggregates of lipid molecules. Lipofectamine (lipofectamine) -nucleic acid complexes are also contemplated.

Whether or not the method is used to introduce an exogenous nucleic acid into a host cell or expose a cell to an inhibitor of the invention, a variety of assays may be performed in order to confirm the presence of recombinant DNA sequences in the host cell. Such assays include, for example, "molecular biological assays" well known to those skilled in the art, such as southern and northern blots, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, for example, identify substances within the scope of the invention by immunological means (ELISA and Western blotting) or by the assays described herein.

The invention also provides a vector comprising a nucleic acid molecule encoding a CAR. In one aspect, the CAR vector can be directly transduced into a cell, e.g., an immune effector cell, e.g., a T cell or NK cell. In one aspect, the vector is a cloning vector or an expression vector, e.g., a vector, including but not limited to one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, microcarriers, double minichromosomes), retroviral vector constructs, and lentiviral vector constructs. In one aspect, the vector is capable of expressing the CAR construct in a mammalian T cell. In one aspect, the mammalian T cell is a human T cell.

Cell source

Prior to expansion and genetic or other modification, a source of cells (e.g., immune effector cells, e.g., T cells or NK cells) is obtained from the subject. The term "subject" is intended to include living organisms (e.g., mammals) that can elicit an immune response. Examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors.

In certain aspects of the invention, any number of immune effector cell (e.g., T cell or NK cell) lines available in the art may be used. In certain aspects of the invention, any of a variety of techniques known to the skilled artisan (e.g., Ficoll) may be used^TMIsolation) of T cells from a blood component collected from a subject. In a preferred aspect, the obtained sample is obtained from an individual by apheresisCells that circulate blood. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, cells collected by apheresis may be washed to remove plasma fractions and to place the cells in an appropriate buffer or culture medium for subsequent processing steps. In one aspect of the invention, cells are washed with Phosphate Buffered Saline (PBS). In an alternative aspect, the wash liquor lacks calcium and may lack magnesium or may lack many, if not all, divalent cations.

An initial activation step in the absence of calcium may result in amplified activation. As one of ordinary skill in the art will readily appreciate, the washing step can be accomplished by methods known to those of skill in the art, such as by using a semi-automated "flow through" centrifuge (e.g., Cobe 2991 Cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte a, or other saline solutions with or without buffers. Alternatively, the unwanted components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

It is recognized that the methods of the present application can utilize media conditions comprising 5% or less (e.g., 2%) human AB Serum, and utilize known media conditions and combinations, for example, as described in Smith et al, "Ex vivo expansion of human T cells for adaptive immunological use of the novel Xeno-free CTS Immune Cell Serum Replacement" Clinical & Translational immunological (2015)4, e 31; 10.1038/ct.2014.31.

In one aspect, by lysing erythrocytes and depleting monocytes, e.g., by PERCOLL ^TMT cells are isolated from peripheral blood lymphocytes by gradient centrifugation or by countercurrent centrifugal elutriation.

Specific T cell subsets, such as CD3+, CD28+, CD4+, CD8+, CD45RA +, and CD45RO + T cells, can be further isolated by positive or negative selection techniques. For example, in one aspect, byBeads (e.g., 3x28) conjugated with anti-CD 3/anti-CD 28 (e.g., 3x28)

M-450 CD3/CD 28T) for a time sufficient to positively select the desired T cells, and isolating the T cells. In one aspect, the time period is about 30 minutes. In yet another aspect, the time period ranges from 30 minutes to 36 hours or more and all integer values in between. In yet another aspect, the period of time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the period of time is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times can be used to isolate T cells in any situation where there are small numbers of T cells, e.g., as compared to other cell types, e.g., for isolating Tumor Infiltrating Lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. In addition, the efficiency of capturing CD8+ T cells can be increased using longer incubation times. Thus, by simply shortening or extending this time, allowing T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the bead-to-T cell ratio (as further described herein), T cell subsets can be preferentially selected at the beginning of the culture or at other time points during the culture process. Additionally, by increasing or decreasing the ratio of anti-CD 3 and/or anti-CD 28 antibodies on the bead or other surface, T cell subsets can be preferentially selected at the beginning of culture or at other desired time points. The skilled person will appreciate that multiple rounds of selection may also be used in the context of the present invention. In certain aspects, it may be desirable to perform a selection procedure and use "unselected" cells during activation and expansion. "unselected" cells may also receive further rounds of selection.

Enrichment of a population of T cells can be accomplished by a negative selection process with a combination of antibodies directed against surface markers unique to negatively selected cells. One method is to sort and/or select cells by means of negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD 8. In certain aspects, it may be desirable to enrich for or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR +, and FoxP3 +. Alternatively, in certain aspects, regulatory T cells are depleted by anti-C25 conjugated beads or other similar selection methods.

The methods described herein can include selecting a specific immune effector cell (e.g., T cell) subpopulation that is a regulatory T cell-depleted population, CD25+ depleted cells, for example, using negative selection techniques (e.g., described herein). Preferably, the population of regulatory T cell-depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells.

In one embodiment, the regulatory T cells, e.g., CD25+ T cells, are removed from the population using an anti-CD 25 antibody or fragment thereof or a CD25 binding ligand (IL-2). In one embodiment, the anti-CD 25 antibody or fragment thereof or CD 25-binding ligand is conjugated to or coated on a substrate (e.g., a bead). In one embodiment, the anti-CD 25 antibody or fragment thereof is conjugated to a substrate as described herein.

In one embodiment, the compound from Miltenyi is used^TMThe CD25 depleting agent removes regulatory T cells, e.g., CD25+ T cells, from the population. In one embodiment, the ratio of cells to CD25 depleting agent is 1e7 cells to 20uL, or 1e7 cells to 15uL, or 1e7 cells to 10uL, or 1e7 cells to 5uL, or 1e7 cells to 2.5uL, or 1e7 cells to 1.25uL agent. In one embodiment, for example, for depleting regulatory T cells, e.g., CD25+, greater than 500 million cells/ml are used. In yet another aspect, cell concentrations of 600, 700, 800, or 900 million cells/ml are used.

In one embodiment, the population of immune effector cells to be depleted comprises about 6x10⁹And (3) CD25+ T cells. In other aspects, the population of immune effector cells to be depleted comprises about 1x10 ⁹To 1x10¹⁰CD25+ T cells and any betweenAnd the integral value of CD25+ T cells. In one embodiment, the resulting regulatory T cell depleted population has 2x10⁹Regulatory T cells, e.g., CD25+ cells, or less (e.g., 1X 10)⁹、5x10⁸、1x10⁸、5x10⁷、1x10⁷One or fewer CD25+ cells).

In one embodiment, regulatory T cells, e.g., CD25+ cells, are removed from the population using the CliniMAC system with a depletion line kit, e.g., line 162-01. In one embodiment, the CliniMAC system operates on a DEPLETION setting (such as, for example, DEPLETION 2.1).

Without wishing to be bound by a particular theory, reducing the level of negative regulators of immune cells in a subject (e.g., reducing unwanted immune cells (e.g., T) prior to apheresis or during the generation of a CAR-expressing cell product_REGCells) can reduce the risk of relapse in a subject. For example, exhaustion of T_REGMethods of cell culture are known in the art. Reduction of T_REGMethods of cell include, but are not limited to, cyclophosphamide, anti-GITR antibodies (described herein), CD25 depletion, and combinations thereof.

In some embodiments, the method of production comprises reducing T prior to (e.g., depleting) the CAR-expressing cell _REGThe number of cells. For example, the production method comprises contacting the sample (e.g., an apheresis sample) with an anti-GITR antibody and/or an anti-CD 25 antibody (or fragment thereof, or CD 25-binding ligand), e.g., to deplete T cells (e.g., T cells, NK cells) prior to production of a CAR-expressing cell (e.g., T cells, NK cells)_REGA cell.

In one embodiment, the subject is administered a T-reduction prior to harvesting the cells to produce a CAR-expressing cell product_REGOne or more therapies of the cell are pre-treated, thereby reducing the risk of relapse in the subject relative to a CAR-expressing cell therapy. In one embodiment, T is decreased_REGMethods of cell include, but are not limited to, administering to a subject one or more of: cyclophosphamide, an anti-GITR antibody, CD25 depleted, or a combination thereof. Administration of cyclophosphamide, anti-GITR antibodies, CD25 depletion, or a combination thereofOne or more of the can be performed before, during, or after infusion of the CAR-expressing cell product.

In one embodiment, the subject is pre-treated with cyclophosphamide prior to harvesting the cells to produce the CAR-expressing cell product, thereby reducing the risk of relapse in the subject relative to CAR-expressing cell therapy. In one embodiment, the subject is pre-treated with an anti-GITR antibody prior to harvesting the cells to produce the CAR-expressing cell product, thereby reducing the risk of relapse in the subject relative to CAR-expressing cell therapy.

In one embodiment, the population of cells to be removed are neither regulatory T cells nor tumor cells, but are cells that adversely affect the expansion and/or function of CART cells, e.g., cells that express CD14, CD11b, CD33, CD15, or other markers expressed by potential immunosuppressive cells. In one embodiment, it is contemplated that such cells are removed simultaneously with regulatory T cells and/or tumor cells, or after said depletion or in another order.

The methods described herein may include more than one selection step, e.g., more than one depletion step. Enrichment of a population of T cells can be accomplished by a negative selection process, for example, with a combination of antibodies directed against surface markers unique to negatively selected cells. One method is to sort and/or select cells by means of negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail may comprise antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD 8.

The methods described herein can further include removing cells from a population expressing a tumor antigen (e.g., a tumor antigen that does not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14, or CD11b), with the intent to thereby provide a population suitable for regulatory T cell depletion (e.g., CD25+ depleted cells and tumor antigen depleted cells) expressing a CAR (e.g., a CAR described herein). In one embodiment, cells expressing tumor antigens are removed simultaneously with regulatory T cells (e.g., CD25+ cells). For example, the anti-CD 25 antibody or fragment thereof and the anti-tumor antigen antibody or fragment thereof can be attached to the same substrate (e.g., beads) that can be used to remove cells, or the anti-CD 25 antibody or fragment thereof or the anti-tumor antigen antibody or fragment thereof can be attached to separate beads, a mixture of which can be used to remove cells. In other embodiments, the removal of regulatory T cells (e.g., CD25+ cells) and the removal of cells expressing tumor antigens are performed sequentially, and may be performed, for example, in any order.

These methods are also provided, the methods comprising removing cells expressing checkpoint inhibitory proteins (e.g., the checkpoint inhibitory proteins described herein), e.g., one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells, from a population, thereby providing a population of regulatory T cell-depleted (e.g., CD25+ depleted cells) and checkpoint inhibitory protein-depleted cells (e.g., PD1+, LAG3+, and/or TIM3+ depleted cells). Examples of exemplary checkpoint inhibitors include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR β. In one embodiment, cells expressing checkpoint inhibitory proteins are removed simultaneously with regulatory T cells (e.g., CD25+ cells). For example, an anti-CD 25 antibody or fragment thereof and an anti-checkpoint inhibitory protein antibody or fragment thereof can be linked to the same beads that can be used to remove cells, or an anti-CD 25 antibody or fragment thereof or an anti-checkpoint inhibitory protein antibody or fragment thereof can be linked to separate beads, a mixture of which can be used to remove cells. In other embodiments, removal of regulatory T cells (e.g., CD25+ cells) and removal of cells expressing checkpoint inhibitory proteins are performed sequentially and may be performed, for example, in any order.

In one embodiment, a population of T cells may be selected that express one or more of: IFN-gamma, TNF alpha, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B and perforin, or other suitable molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, for example, by the methods described in PCT publication No. WO 2013/126712.

The concentration of cells and surfaces (e.g., particles, such as beads) can be varied in order to isolate a desired cell population by positive or negative selection. In certain aspects, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (e.g., increase the cell concentration) to ensure maximum contact between the cells and the beads. For example, in one aspect, a concentration of 20 hundred million cells/ml is used. In one aspect, a concentration of 10 hundred million cells/ml is used. In yet another aspect, greater than 1 hundred million cells/ml is used. In yet another aspect, a cell concentration of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 million cells/ml is used. In yet another aspect, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/ml is used. In other aspects, concentrations of 1.25 or 1.5 billion cells/ml can be used.

The use of high concentrations can result in increased cell yield, cell activation and cell expansion. In addition, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest (e.g., CD28 negative T cells), or cells from samples in which many tumor cells are present (e.g., leukemia blood, tumor tissue, etc.). Such cell populations may have therapeutic value and would be desirable to obtain. For example, the use of high concentrations of cells allows for more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

In a related aspect, it may be desirable to use lower cell concentrations. By greatly diluting the mixture of T cells and surfaces (e.g., particles such as beads), particle-cell interactions are minimized. This selects cells expressing a high amount of the desired antigen to be bound to the particle. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells at diluted concentrations. In one aspect, the cell concentration used is 5 × 10e 6/ml. In other aspects, the concentration used may be about 1x10⁵From ml to 1X10⁶Ml and any integer value therebetween.

In other aspects, the cells can be incubated at 2-10 ℃ or room temperature on a shaker at different speeds for different lengths of time.

T cells for stimulation may also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing fluid. Although many freezing fluids and parameters are known in the art and will be used in this context, one method involves the use of PBS containing 20% DMSO and 8% human serum albumin, or a medium containing 10

% dextran

40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-a, 31.25% dextrose 5%, 0.45% NaCl, 10

% dextran

40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell-free medium such as one containing Hespan and Plasmalyte a, the cells are then frozen to-80 ℃ at a rate of 1 °/minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods may also be used, as well as immediate uncontrolled freezing at-20 ℃ or in liquid nitrogen.

In certain aspects, cryopreserved cells are thawed and washed as described herein and allowed to stand at room temperature for 1 hour prior to activation using the methods of the invention.

It is also contemplated in the context of the present invention that a blood sample or apheresis product is collected from a subject at some time period prior to when expanded cells as described herein may be needed. In this regard, the cell source to be expanded can be collected at any point in time as desired, and the desired cells (such as immune effector cells, e.g., T cells or NK cells) can be isolated and frozen for later use in cell therapy (e.g., T cell therapy) for any kind of disease or condition (such as those described herein) that would benefit from cell therapy (e.g., T cell therapy). In one aspect, the blood sample or apheresis is taken from a generally healthy subject. In certain aspects, a blood sample or apheresis is taken from a generally healthy subject at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain aspects, immune effector cells (e.g., T cells or NK cells) can be expanded, frozen, and used at a later time. In certain aspects, the sample is taken from a patient shortly after diagnosis of a particular disease as described herein, but prior to any treatment. In yet another aspect, cells are isolated from a blood sample or apheresis of a subject prior to any number of relevant treatment modalities, including, but not limited to, drug therapy, such as natalizumab, efuzumab, antiviral drugs, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies, or other immune scavengers such as CAMPATH, anti-CD 3 antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.

In yet another aspect of the invention, the T cells are obtained from the subject immediately after leaving the subject with treatment for functional T cells. In this respect, it has been observed that after certain cancer treatments, in particular with drugs that damage the immune system, the quality of the T cells obtained may be optimal or improved in terms of their ex vivo expansion capacity during the time shortly after the treatment when the patient will normally recover from the treatment. Likewise, these cells can be in a preferred state of enhanced engraftment and in vivo expansion after being manipulated ex vivo using the methods described herein. It is therefore contemplated in the context of the present invention that blood cells, including T cells, dendritic cells or other cells of the hematopoietic lineage, are collected during this recovery phase. In addition, in certain aspects, mobilization (e.g., with GM-CSF) and conditioning regimens can be used to create conditions in a subject in which repopulation, recirculation, regeneration, and/or expansion of a particular cell type is favored, particularly during a defined time window following treatment. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

In one embodiment, the immune effector cells expressing a CAR molecule (e.g., a CAR molecule described herein) are obtained from a subject that has received an immunopotentiating low dose of an mTOR inhibitor. In one embodiment, the population of immune effector cells (e.g., T cells) to be engineered to express the CAR is harvested after a sufficient time for the mTOR inhibitor or after sufficient administration of an immunopotentiating low dose of the mTOR inhibitor, such that the level of PD1 negative immune effector cells (e.g., T cells) or the ratio of PD1 negative immune effector cells (e.g., T cells)/PD 1 positive immune effector cells (e.g., T cells) in or harvested from the subject has been at least transiently increased.

In other embodiments, a population of immune effector cells (e.g., T cells) that have been or will be engineered to express a CAR can be treated ex vivo by contacting with an mTOR inhibitor in an amount that increases the number of PD1 negative immune effector cells (e.g., T cells) or increases the ratio of PD1 negative immune effector cells (e.g., T cells)/PD 1 positive immune effector cells (e.g., T cells).

In one embodiment, the population of T cells is diacylglycerol kinase (DGK) deficient. DGK-deficient cells include cells that do not express DGK RNA or protein or have reduced or inhibited DGK activity. DGK-deficient cells can be generated by genetic means, e.g., administration of an RNA interfering agent (e.g., siRNA, shRNA, miRNA) that reduces or prevents DGK expression. Alternatively, DGK-deficient cells can be produced by treatment with a DGK inhibitor as described herein.

In one embodiment, the population of T cells is ikros deficient. Ikaro-deficient cells include cells that do not express Ikaros RNA or protein or have reduced or inhibited Ikaro activity, and Ikaro-deficient cells can be generated by genetic methods, e.g., administration of RNA interference agents (e.g., siRNA, shRNA, miRNA) that reduce or prevent expression of Ikaro. Alternatively, ikros-deficient cells can be generated by treatment with an ikros inhibitor (e.g., lenalidomide).

In embodiments, the population of T cells is DGK-deficient and Ikaros-deficient, e.g., does not express DGK and Ikaros, or has reduced or inhibited DGK activity and Ikaros activity. Such DGK and Ikaros deficient cells can be produced by any of the methods described herein.

In one embodiment, the NK cells are obtained from a subject. In another embodiment, the NK cell is an NK cell line, e.g., an NK-92 cell line (Conkwest).

Allogeneic CAR

In embodiments described herein, the immune effector cell may be an allogeneic immune effector cell, e.g., a T cell or an NK cell. For example, the cells can be allogeneic T cells, e.g., allogeneic T cells lacking functional T Cell Receptors (TCRs) and/or expression of Human Leukocyte Antigens (HLA) (e.g., HLA class I and/or HLA class II).

A T cell lacking a functional TCR may, for example, be engineered such that it does not express any functional TCR on its surface; engineered such that it does not express one or more of the subunits that make up a functional TCR (e.g., engineered such that it does not express or exhibits reduced expression of TCR α, TCR β, TCR γ, TCR δ, TCR ∈ and/or TCR ζ); or engineered so that it produces very little functional TCR on its surface. Alternatively, T cells may express a largely impaired TCR, for example, by expressing a mutated or truncated form of one or more TCR subunits. The term "substantially impaired TCR" means that such TCR will not elicit an adverse immune response in the host.

The T cell described herein may, for example, be engineered such that it does not express a functional HLA on its surface. For example, the T cells described herein can be engineered such that cell surface expression of HLA (e.g., HLA class I and/or HLA class II) is down-regulated. In some aspects, down-regulation of HLA can be achieved by reducing or eliminating β -2 microglobulin (B2M) expression.

In some embodiments, the T cell may lack a functional TCR and a functional HLA, e.g., HLA class I and/or HLA class II.

Modified T cells lacking functional TCR and/or HLA expression can be obtained by any suitable means, including knockout or knock-down of one or more subunits of TCR or HLA. For example, T cells can include TCRs and/or HLA knocked down using sirnas, shrnas, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), transcription activator-like effector nucleases (TALENs), or zinc finger endonucleases (ZFNs).

In some embodiments, the allogeneic cells may be cells that do not express or express low levels of inhibitory molecules, e.g., cells engineered by any of the methods described herein. For example, the cell can be a cell that does not express or expresses at low levels an inhibitory molecule, e.g., a cell that can reduce the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR β. Inhibition of the inhibitory molecule (e.g., by inhibition at the DNA, RNA, or protein level) can optimize the performance of the cell expressing the CAR. In embodiments, inhibitory nucleic acids, e.g., dsrnas, e.g., sirnas or shrnas, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), transcription activator-like effector nucleases (TALENs), or zinc finger endonucleases (ZFNs), e.g., as described herein, can be used.

siRNA and shRNA for inhibiting TCR or HLA

In some embodiments, TCR expression and/or HLA expression may be inhibited in a T cell using an siRNA or shRNA targeting a nucleic acid encoding a TCR and/or HLA and/or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR β).

Expression of the siRNA and shRNA in T cells can be achieved using any conventional expression system (e.g., such as a lentiviral expression system).

Exemplary shrnas that down-regulate the expression of components of a TCR are described, for example, in U.S. publication No. 2012/0321667. Exemplary sirnas and shrnas that down-regulate expression of HLA class I genes and/or HLA class II genes are described, for example, in U.S. publication No. US 2007/0036773.

CRISPR inhibiting TCR or HLA

As used herein, "CRISPR" or "CRISPR against TCR and/or HLA" or "CRISPR inhibiting TCR and/or HLA" refers to a set of clustered regularly interspaced short palindromic repeats or a system comprising such a set of repeats. As used herein, "Cas" refers to a CRISPR-associated protein. The CRISPR/Cas "system refers to a system derived from CRISPR and Cas that can be used to silence or mutate TCR and/or HLA genes and/or inhibitory molecules described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR β).

The naturally occurring CRISPR/Cas system was found in approximately 40% sequenced eubacterial genomes and 90% sequenced archaea. Grissa et al (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al (2007) Science 315: 1709-1712; marragini et al (2008) Science 322: 1843-1845.

The CRISPR/Cas system has been modified for gene editing (silencing, enhancing or altering specific genes) in eukaryotes such as mice or primates. Wiedenheft et al (2012) Nature 482: 331-8. This is achieved by introducing into eukaryotic cells a plasmid containing a specifically designed CRISPR and one or more suitable Cas.

CRISPR sequences, sometimes referred to as CRISPR loci, comprise alternating repeats and spacers. In naturally occurring CRISPRs, the spacer sequence typically comprises a sequence that is foreign to the bacterium, such as a plasmid or phage sequence; in the TCR and/or HLA CRISPR/Cas systems, the spacer sequence is derived from a TCR or HLA gene sequence.

RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs. These comprise a spacer sequence flanked by repeated sequences. RNA guides other Cas proteins silence exogenous genetic elements at the RNA or DNA level. Horvath et al (2010) Science 327: 167-170; makarova et al (2006) Biology Direct 1: 7. The spacer sequence thus serves as a template for the RNA molecule, similar to siRNA. Pennisi (2013) Science 341: 833-836.

As these occur naturally in many different types of bacteria, the exact layout of CRISPR and the structure, function and number of Cas genes and products vary more or less between species. Haft et al (2005) PLoS Compout.biol.1: e 60; kunin et al (2007) Genome biol.8: R61; mojica et al (2005) J.mol.Evol.60: 174-182; bolotin et al (2005) Microbiol.151: 2551-2561; pourcel et al (2005) Microbiol.151: 653-663; and Stern et al (2010) trends. Genet.28: 335-340. For example, the Cse (Cas subtype, e.g., escherichia coli) protein (e.g., CasA) forms a functional complex Cascade that processes the CRISPR RNA transcript into a Cascade-retaining spacer-repeat unit. Brouns et al (2008) Science 321: 960-964. In other prokaryotes, Cas6 processes CRISPR transcripts. CRISPR-based phage inactivation in e.coli requires Cascade and Cas3, but not Cas1 or Cas 2. The Cmr (Cas RAMP module) proteins in Pyrococcus furiosus (Pyrococcus furiosus) and other prokaryotes form functional complexes with small CRISPR RNA that recognize and cleave complementary target RNA. A simpler CRISPR system relies on a Cas9 protein, a nuclease with two effective cleavage sites, one for each strand of the double helix. Cas9 and modified CRISPR locus RNA combinations can be used in a system for editing genes. Pennisi (2013) Science 341: 833-836.

The CRISPR/Cas system can thus be used to edit TCR and/or HLA genes (add or delete base pairs), or cause premature cessation, which thus reduces expression of TCR and/or HLA. The CRISPR/Cas system can alternatively be used as RNA interference, turning off TCR and/or HLA genes in a reversible manner. In mammalian cells, for example, RNA can direct Cas protein to TCR and/or HLA promoters, spatially blocking RNA polymerase.

Artificial CRISPR/Cas systems that inhibit TCR and/or HLA can be generated using techniques known in the art, for example, those described in U.S. publication No. 20140068797 and Cong (2013) Science 339: 819-823. Other artificial CRISPR/Cas systems known in the art to inhibit TCR and/or HLA can also be generated, for example, as described in Tsai (2014) Nature biotechnol, 32: 6569-576; U.S. patent nos. 8,871,445; 8,865,406, respectively; 8,795,965, respectively; 8,771,945 and 8,697,359.

TALEN inhibiting TCR and/or HLA

"TALEN" or "TALEN against HLA and/or TCR" or "TALEN inhibiting HLA and/or TCR" refers to a transcriptional activator-like effector nuclease, an artificial nuclease that can be used to edit HLA and/or TCR genes and/or inhibitory molecules described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (tnfrf 14 or CD270), KIR, A2aR, class I, MHC class II, GAL9, adenosine fr β).

TALENs were artificially generated by fusion of the TAL effector DNA binding domain to the DNA cleavage domain. Transcription activator-like effects (TALEs) can be engineered to bind any DNA sequence of interest, including a portion of an HLA or TCR gene. By combining engineered TALEs with DNA cleavage domains, restriction enzymes specific for any DNA sequence of interest (including HLA or TCR sequences) can be generated. These enzymes can then be introduced into cells where they can be used for genome editing. Boch (2011) Nature biotech.29: 135-6; and Boch et al (2009) Science 326: 1509-12; moscou et al (2009) Science 326: 3501.

TALEs are proteins secreted by bacteria of the genus Xanthomonas (Xanthomonas). The DNA binding domain contains a repetitive highly conserved 33-34 amino acid sequence with the exception of amino acids 12 and 13. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition. They can thus be engineered to bind to a DNA sequence of interest.

To generate TALENs, TALE proteins are fused to a nuclease (N), which is a wild-type or mutant fokl endonuclease. Several mutations have been made to fokl in order for it to be used in TALENs; these mutations improve, for example, cleavage specificity or activity. Cerak et al (2011) Nucl. acids Res.39: e 82; miller et al (2011) Nature Biotech.29: 143-8; hockemeyer et al (2011) Nature Biotech.29: 731-734; wood et al (2011) Science 333: 307; doyon et al (2010) Nature Methods 8: 74-79; szczepek et al (2007) Nature Biotech.25: 786-793; and Guo et al (2010) j.mol.biol.200: 96.

The FokI domain functions as a dimer, thus requiring two constructs with unique DNA binding domains to sites in the target genome that are correctly oriented and spaced. The number of amino acid residues between the TALE DNA binding domain and the fokl cleavage domain and the number of bases between two independent TALEN binding sites appear to be both important parameters for achieving high levels of activity. Miller et al, (2011) Nature Biotech.29: 143-8.

HLA or TCR TALENs can be used inside cells to create double-strand breaks (DSBs). Mutations can be introduced at the fracture site if the repair mechanism improperly repairs the fracture via non-homologous end joining. For example, inappropriate repair may introduce frameshift mutations. Alternatively, the foreign DNA can be introduced into the cell with the TALEN; depending on the sequence of the foreign DNA and chromosomal sequence, this approach can be used to correct a defect in the HLA or TCR gene or to introduce such a defect into the wt HLA or TCR gene, thus reducing expression of HLA or TCR.

TALENs specific for sequences in HLA or TCR can be constructed using any method known in the art, including various schemes using modular components. Zhang et al (2011) Nature Biotech.29: 149-53; geibler et al (2011) PLoS ONE 6: e 19509.

Zinc finger nucleases for inhibition of HLA and/or TCR

"ZFN" or "zinc finger nuclease" or "ZFN against HLA and/or TCR" or "HLA and/or TCR-inhibiting ZFN" refers to a zinc finger nuclease, which is an artificial nuclease that can be used to edit HLA and/or TCR genes and/or inhibitory molecules described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL fr 9, adenosine nuclease, and tgp).

Like TALENs, ZFNs comprise a FokI nuclease domain (or derivative thereof) fused to a DNA binding domain. In the case of ZFNs, the DNA binding domain comprises one or more zinc fingers. Carroll et al (2011) Genetics Society of America 188: 773-782; and Kim et al (1996) Proc.Natl.Acad.Sci.USA 93: 1156-1160.

Zinc fingers are small protein structural motifs stabilized by one or more zinc ions. The zinc finger may, for example, comprise Cys₂His₂And can recognize about 3bp sequence. A variety of zinc fingers of known specificity can be combined to produce a multi-finger polypeptide that recognizes a sequence of about 6, 9, 12, 15, or 18 bp. A variety of selection and modular assembly techniques can be used to generate zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast single-hybrid systems, bacterial single-hybrid and two-hybrid systems, and mammalian cells.

Like TALENs, ZFNs must dimerize to cleave DNA. Therefore, a pair of ZFNs is required to target non-palindromic DNA sites. Two independent ZFNs must bind opposite strands of DNA with their nucleases properly spaced apart. Bitinaite et al, (1998) Proc. Natl. Acad. Sci. USA95: 10570-5.

Also like TALENs, ZFNs can create double-stranded breaks in DNA that, if improperly repaired, can produce frameshift mutations, resulting in reduced expression and amount of HLA and/or TCR in the cell. ZFNs can also be used with homologous recombination to mutate in HLA or TCR genes.

ZFNs specific for sequences in HLA and/or TCR can be constructed using any method known in the art. See, e.g., Provasi (2011) Nature Med.18: 807-815; torikai (2013) Blood 122: 1341-1349; cathomen et al (2008) mol. Ther.16: 1200-7; guo et al (2010) j.mol.biol.400: 96; U.S. patent publication 2011/0158957; and U.S. patent publication 2012/0060230.

Telomerase expression

While not wishing to be bound by any particular theory, in some embodiments, the therapeutic T cells have a short-term persistence in the patient due to telomere shortening in the T cells; thus, transfection with a telomerase gene can extend telomeres of T cells and improve the persistence of T cells in a patient. See Carl June, "adaptive T cell therapy for cancer in the Clinical", Journal of Clinical Investigation,117:1466-1476 (2007). Thus, in one embodiment, an immune effector cell (e.g., a T cell) ectopically expresses a telomerase subunit, e.g., a catalytic subunit of telomerase (e.g., TERT, e.g., hTERT). In some aspects, the disclosure provides a method of generating a CAR-expressing cell, comprising contacting the cell with a nucleic acid encoding a telomerase subunit (e.g., a catalytic subunit of telomerase (e.g., TERT, e.g., hTERT)). The cell can be contacted with the nucleic acid prior to, concurrently with, or after contacting the construct encoding the CAR.

In one aspect, the disclosure features a method of generating a population of immune effector cells (e.g., T cells, NK cells). In one embodiment, the method comprises: providing a population of immune effector cells (e.g., T cells or NK cells), contacting the population of immune effector cells with a nucleic acid encoding a CAR; and contacting a population of immune effector cells with a nucleic acid encoding a telomerase subunit (e.g., hTERT) under conditions that allow for expression of the CAR and telomerase.

In one embodiment, the nucleic acid encoding a telomerase subunit is DNA. In one embodiment, the nucleic acid encoding a telomerase subunit comprises a promoter capable of driving expression of the telomerase subunit.

In one embodiment, hTERT has the amino acid sequence of the GenBank protein IDAAC51724.1 (Meyerson et al, "hEST 2, the pure Human temporal Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during immunization" Cell Vol.90, No. 4, 1997, 8/22, p.785-795):

MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRGCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD(SEQ ID NO:157)

in one embodiment, the hTERT has a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 157. In one embodiment, hTERT has the sequence of SEQ ID NO: 157. In one embodiment, hTERT comprises a deletion (e.g., of no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both. In one embodiment, hTERT comprises a transgenic amino acid sequence (e.g., a transgenic amino acid sequence of no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both.

In one embodiment, hTERT Is encoded by the following nucleic acid sequence of GenBank accession No. AF018167 (Meyerson et al, "hEST 2, the pure Human temporal Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and duringImmobilisation" Cell Vol.90, No. 4, 1997, 8/22, pp.785-795):

1 caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcgatgc

61 cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactac cgcgaggtgc

121 tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg gtgcagcgcg

181 gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg ccctgggacg

241 cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag gagctggtgg

301 cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc ttcggcttcg

361 cgctgctgga cggggcccgc gggggccccc ccgaggcctt caccaccagc gtgcgcagct

421 acctgcccaa cacggtgacc gacgcactgc gggggagcgg ggcgtggggg ctgctgttgc

481 gccgcgtggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc tttgtgctgg

541 tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca

601 ctcaggcccg gcccccgcca cacgctagtg gaccccgaag gcgtctggga tgcgaacggg

661 cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagcc ccgggtgcga

721 ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggccc aggcgtggcg

781 ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccac ccgggcagga

841 cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacc tgccagaccc gccgaagaag

901 ccacctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc

961 agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac acgccttgtc

1021 ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaag gagcagctgc

1081 ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcgg aggctcgtgg

1141 agaccatctt tctgggttcc aggccctgga tgccagggac tccccgcagg ttgccccgcc

1201 tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc

1261 agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg gtcaccccag

1321 cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggccccc gaggaggagg

1381 acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcagcccc tggcaggtgt

1441 acggcttcgt gcgggcctgc ctgcgccggc tggtgccccc aggcctctgg ggctccaggc

1501 acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctgggg aagcatgcca

1561 agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct tggctgcgca

1621 ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag gagatcctgg

1681 ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcagg tctttctttt

1741 atgtcacgga gaccacgttt caaaagaaca ggctcttttt ctaccggaag agtgtctgga

1801 gcaagttgca aagcattgga atcagacagc acttgaagag ggtgcagctg cgggagctgt

1861 cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacg tccagactcc

1921 gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactac gtcgtgggag

1981 ccagaacgtt ccgcagagaa aagagggccg agcgtctcac ctcgagggtg aaggcactgt

2041 tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc tctgtgctgg

2101 gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg gcccaggacc

2161 cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac accatccccc

2221 aggacaggct cacggaggtc atcgccagca tcatcaaacc ccagaacacg tactgcgtgc

2281 gtcggtatgc cgtggtccag aaggccgccc atgggcacgt ccgcaaggcc ttcaagagcc

2341 acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct cacctgcagg

2401 agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg aatgaggcca

2461 gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg cgcatcaggg

2521 gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctcc acgctgctct

2581 gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcgg cgggacgggc

2641 tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa

2701 ccttcctcag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga

2761 agacagtggt gaacttccct gtagaagacg aggccctggg tggcacggct tttgttcaga

2821 tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccgg accctggagg

2881 tgcagagcga ctactccagc tatgcccgga cctccatcag agccagtctc accttcaacc

2941 gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tggggtcttg cggctgaagt

3001 gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgc accaacatct

3061 acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag ctcccatttc

3121 atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgac acggcctccc

3181 tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggcc aagggcgccg

3241 ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattc ctgctcaagc

3301 tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggaca gcccagacgc

3361 agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca gccaacccgg

3421 cactgccctc agacttcaag accatcctgg actgatggcc acccgcccac agccaggccg

3481 agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagg gaggggcggc

3541 ccacacccag gcccgcaccg ctgggagtct gaggcctgag tgagtgtttg gccgaggcct

3601 gcatgtccgg ctgaaggctg agtgtccggc tgaggcctga gcgagtgtcc agccaagggc

3661 tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcg gctccacccc

3721 agggccagct tttcctcacc aggagcccgg cttccactcc ccacatagga atagtccatc

3781 cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccac ccccaccatc

3841 caggtggaga ccctgagaag gaccctggga gctctgggaa tttggagtga ccaaaggtgt

3901 gccctgtaca caggcgagga ccctgcacct ggatgggggt ccctgtgggt caaattgggg

3961 ggaggtgctg tgggagtaaa atactgaata tatgagtttt tcagttttga aaaaaaaaaa

4021 aaaaaaa(SEQ ID NO:158)

in one embodiment, the hTERT is encoded by a nucleic acid having a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO. 158. In one embodiment, the hTERT is encoded by the nucleic acid of SEQ ID NO. 158.

Activating and expanding T cells

T cells can be activated and expanded generally using methods as described, for example, in the following references: us patent 6,352,694; 6,534,055, respectively; 6,905,680, respectively; 6,692,964, respectively; 5,858,358, respectively; 6,887,466, respectively; 6,905,681, respectively; 7,144,575, respectively; 7,067,318, respectively; 7,172,869, respectively; 7,232,566, respectively; 7,175,843, respectively; 5,883,223, respectively; 6,905,874, respectively; 6,797,514, respectively; 6,867,041, respectively; and U.S. patent application publication No. 20060121005.

In general, the T cells of the invention can be expanded by contacting a surface that has been linked to an agent that stimulates a signal associated with the CD3/TCR complex and a ligand that stimulates a costimulatory molecule on the surface of the T cell. In particular, the population of T cells can be stimulated as described herein, such as by contact with an anti-CD 3 antibody or antigen-binding fragment thereof or an anti-CD 2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryozoalactone) in conjunction with a calcium ionophore. To co-stimulate accessory molecules on the surface of T cells, ligands that bind the accessory molecules are used. For example, the T cell population may be adapted to stimulate T Under conditions in which the cells proliferate, with an anti-CD 3 antibody and an anti-CD 28 antibody. To stimulate proliferation of CD4+ T cells or CD8+ T cells, anti-CD 3 antibodies and anti-CD 28 antibodies may be used. Examples of anti-CD 28 antibodies include 9.3, B-T3, XR-CD28(Diaclone,

france), as other methods known in the art (Berg et al, Transplant Proc.30(8):3975-3977, 1998; haanen et al, J.exp.Med.190(9):13191328,1999; garland et al, J.Immunol meth.227(1-2):53-63,1999).

In certain aspects, the primary and costimulatory signals for T cells can be provided by different protocols. For example, the substance providing each signal may be in solution or coupled to a surface. When coupled to a surface, a species may be coupled to the same surface (i.e., in "cis" form) or to a separate surface (i.e., in "trans" form). Alternatively, one species may be coupled to the surface and the other species in solution. In one aspect, the substance that provides the co-stimulatory signal is bound to the cell surface and the substance that provides the primary activation signal is in solution or coupled to the surface. In certain aspects, both substances may be in solution. In one aspect, the substance may be in a soluble form and subsequently crosslinked to a surface, such as a cell expressing an Fc receptor or antibody or other conjugate that will bind to the substance. In this regard, the present invention contemplates artificial antigen presenting cells (aapcs) for activating and expanding T cells, see, e.g., U.S. patent application publication nos. 20040101519 and 20060034810.

In one aspect, both substances are immobilized on a bead, wherein they are immobilized on the same bead, i.e., "cis", or to separate beads, i.e., "trans". For example, the agent that provides a primary activation signal is an anti-CD 3 antibody or antigen-binding fragment thereof and the agent that provides a co-stimulatory signal is an anti-CD 28 antibody or antigen-binding fragment thereof; and both substances are co-immobilized to the same bead with equivalent molecular numbers. In one aspect, a 1:1 ratio of each antibody bound to the beads is used for CD4+ T cell expansion and T cell growth. In certain aspects of the invention, the ratio of anti-CD 3: CD28 antibody bound to beads is used such that increased T cell expansion is observed as compared to the expansion observed when using the ratio 1: 1. In one particular aspect, an increase of about 1-fold to about 3-fold is observed as compared to the expansion observed when using the ratio of 1: 1. In one aspect, the ratio of CD3 to CD28 antibody bound to the beads is from 100:1 to 1:100 and all integer values therebetween. In one aspect of the invention, more anti-CD 28 antibody binds to the particles than anti-CD 3 antibody, i.e., the ratio of CD3 to CD28 is less than 1. In certain aspects of the invention, the ratio of anti-CD 28 antibody to anti-CD 3 antibody bound to beads is greater than 2: 1. In a particular aspect, a 1:100 to bead bound CD3: CD28 antibody ratio is used. In one aspect, a 1:75 to bead bound CD3: CD28 antibody ratio is used. In yet another aspect, a 1:50 to bead bound CD3: CD28 antibody ratio is used. In one aspect, a 1:30 to bead bound CD3 to CD28 antibody ratio is used. In a preferred aspect, a 1:10 to bead bound CD3 to CD28 antibody ratio is used. In one aspect, a 1:3 to bead bound CD3: CD28 antibody ratio is used. In yet another aspect, a 3:1 to bead bound CD3: CD28 antibody ratio is used.

Particle to cell ratios of 1:500 to 500:1 and any integer value therebetween can be used to stimulate T cells or other target cells. As one of ordinary skill in the art can readily appreciate, the particle-to-cell ratio can depend on the particle size relative to the target cell. For example, small size beads can only bind some cells, while larger beads can bind many cells. In certain aspects, the ratio of cells to particles ranges from 1:100 to 100:1 and any integer value therebetween, and in other aspects, the ratio comprises 1:9 to 9:1 and any integer value therebetween, which can also be used to stimulate T cells. The ratio of anti-CD 3 and anti-CD 28 conjugate particles to T cells that result in T cell stimulation can vary as indicated above, however certain preferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 and 15:1, one preferred ratio being at least 1:1 particles per T cell. In one aspect, a particle to cell ratio of 1:1 or less is used. In a particular aspect, a preferred particle to cell ratio is 1: 5. In other aspects, the particle to cell ratio can vary depending on the date of stimulation. For example, in one aspect, the particle to cell ratio is 1:1 to 10:1 on day 1, and thereafter additional particles are added to the cells daily or every 1 day for up to 10 days, at a final ratio of 1:1 to 1:10 (based on cell count at the time of day of addition). In a particular aspect, the particle to cell ratio is 1:1 on the 1 st stimulation day and is adjusted to 1:5 on the third and fifth stimulation days. In one aspect, the particles are added to a final ratio of 1:1 on a daily or every 1 day basis to the 1 st stimulation day, and to a final ratio of 1:5 on the third and fifth stimulation days. In one aspect, the particle to cell ratio is 2:1 on day 1 stimulation and is adjusted to 1:10 on the third and fifth stimulation days. In one aspect, the particles are added to a final ratio of 1:1 on a daily or every 1 day basis to the 1 st stimulation day, and to a final ratio of 1:10 on the third and fifth stimulation days. Those skilled in the art will appreciate that a variety of other ratios may be suitable for use in the present invention. In particular, the ratio will vary depending on the particle size and cell size and type. In one aspect, the most common ratios used are close to 1:1, 2:1 and 3:1 on day 1.

In other aspects of the invention, cells (e.g., T cells) are combined with the substance-coated beads, the beads and cells are then separated, and the cells are then cultured. In an alternative aspect, the substance-coated beads and cells are not separated prior to culturing, but are cultured together. In yet another aspect, the beads and cells are first concentrated by applying a force (e.g., a magnetic force) that results in increased attachment of cell surface markers, thereby inducing cell stimulation.

For example, cell surface proteins can be attached by contacting T cells with paramagnetic beads (3x28 beads) attached to anti-CD 3 and anti-CD 28. In one aspect, cells (e.g., 10)⁴Is as follows to 10⁹T cells) and beads (e.g.,

m-450 CD3/CD 28T paramagnetic beads in a ratio of 1:1) in slow phaseThe rinses are combined, for example, in PBS (without divalent cations such as calcium and magnesium). Again, one of ordinary skill in the art will readily appreciate that any cell concentration may be used. For example, the target cells may be very rare in the sample and may comprise only 0.01% of the sample or the entire sample (i.e., 100%) may contain the target cells of interest. Thus, any number of cells is within the context of the present invention. In certain aspects, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between the cells and the particles. For example, in one aspect, a concentration of about 100, 90, 80, 70, 60, 50, or 20 hundred million cells/ml is used. In one aspect, greater than 1 hundred million cells/ml is used. In yet another aspect, a cell concentration of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 million cells/ml is used. In yet another aspect, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/ml is used. In other aspects, concentrations of 1.25 or 1.5 billion cells/ml can be used. The use of high concentrations can result in increased cell yield, cell activation and cell expansion. In addition, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28 negative T cells. In certain aspects, such cell populations may have therapeutic value and would be desirable to obtain. For example, the use of high concentrations of cells allows for more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

In one embodiment, a cell transduced with a nucleic acid encoding a CAR (e.g., a CAR described herein) is expanded, e.g., expanded by the methods described herein. In one embodiment, the cells are expanded in culture for several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). In one embodiment, the cells are expanded for a period of 4 to 9 days. In one embodiment, the cells are expanded for a period of 8 days or less (e.g., 7, 6, or 5 days). In one embodiment, cells (e.g., CD33 CAR cells described herein) are expanded in culture for 5 days and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions. Potency may be defined, for example, by various T cell functions such as proliferation, target cell killing, cytokine production, activation, migration, or a combination thereof. In one embodiment, cells expanded for 5 days in culture (e.g., CD33 CAR cells described herein) exhibit at least a 1, 2, 3, or 4 fold increase in cell doubling upon antigen stimulation as compared to the same cells expanded for 9 days in culture under the same culture conditions. In one embodiment, cells expressing a CD33 CAR described herein are expanded in culture for 5 days, and the cells produced exhibit a higher production of proinflammatory cytokines, e.g., a higher level of IFN- γ and/or GM-CSF, compared to the same cells expanded in culture for 9 days under the same culture conditions. In one embodiment, cells expanded for 5 days in culture (e.g., CD33 CAR cells described herein) exhibit at least a 1, 2, 3, 4, 10-fold or more increase in pro-inflammatory cytokine production (e.g., IFN- γ levels and/or GM-CSF levels) in pg/ml as compared to the same cells expanded for 9 days in culture under the same culture conditions.

In one aspect of the invention, the mixture may be incubated for several hours (about 3 hours) to about 14 days or any hour integer value therebetween. In one aspect, the mixture can be cultured for 21 days. In one aspect of the invention, the beads and T cells are cultured together for about 8 days. In one aspect, the beads and T cells are cultured together for 2-3 days. Several rounds of stimulation may also be required so that T cell culture times may be 60 days or longer. Suitable conditions for T cell culture include suitable media (e.g., minimal or RPMI medium 1640 or X-vivo15, (Lonza)) which may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN- γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF β, and TNF α or any other additive known to the skilled artisan for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasma protein powder (plasmanate), and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The culture medium may include RPMI1640, AIM-V, DMEM, MEM, alpha-MEM, F-12X-Vivo15 and X-Vivo20, optizer, together with added amino acids, sodium pyruvate and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or defined group hormones and/or some amount of cytokines sufficient to grow and expand T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures and not in cell cultures to be infused into subjects. The target cells are maintained under conditions necessary to support growth, e.g., a suitable temperature (e.g., 37 ℃) and atmosphere (e.g., air plus 5% CO) ₂)。

In one embodiment, the cells are expanded in a suitable medium (e.g., a medium described herein) comprising one or more interleukins that result in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold, 350-fold) increase in the cells over a 14 day expansion period (e.g., as measured by the methods described herein (e.g., flow cytometry)). In one embodiment, the cells are expanded in the presence of IL-15 and/or IL-7 (e.g., IL-15 and IL-7).

In embodiments, the methods described herein (e.g., methods of producing a CAR-expressing cell) comprise removing regulatory T cells, e.g., CD25+ T cells, from a population of cells, e.g., using an anti-CD 25 antibody or fragment thereof or a CD25 binding ligand, IL-2. Described herein are methods of removing regulatory T cells (e.g., CD25+ T cells) from a population of cells. In embodiments, the method (e.g., production method) further comprises: contacting a population of cells (e.g., a population of cells in which regulatory T cells (such as CD25+ T cells) have been depleted; or a population of cells that have been previously contacted with an anti-CD 25 antibody, fragment thereof, or CD25 binding ligand) with IL-15 and/or IL-7. For example, a population of cells (e.g., a population of cells that have been previously contacted with an anti-CD 25 antibody, fragment thereof, or CD25 binding ligand) is expanded in the presence of IL-15 and/or IL-7.

In some embodiments, a CAR-expressing cell described herein is contacted with a composition comprising an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of an IL-15 polypeptide and an IL-15Ra polypeptide (e.g., hetIL-15) during the production of the CAR-expressing cell (e.g., ex vivo). In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising an IL-15 polypeptide during (e.g., ex vivo) generation of the CAR-expressing cell. In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide during (e.g., ex vivo) production of the CAR-expressing cell. In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising hetIL-15 during the production of the CAR-expressing cell (e.g., ex vivo).

In one embodiment, a CAR-expressing cell described herein is contacted with a composition comprising hetIL-15 during ex vivo expansion. In one embodiment, a CAR-expressing cell described herein is contacted with a composition comprising an IL-15 polypeptide during ex vivo expansion. In one embodiment, a CAR-expressing cell described herein is contacted with a composition comprising both an IL-15 polypeptide and an IL-15Ra polypeptide during ex vivo expansion. In one embodiment, the contacting process results in survival and proliferation of a subpopulation of lymphocytes (e.g., CD8+ T cells).

T cells that have been exposed to varying stimulation times may exhibit different characteristics. For example, common blood or apheresis peripheral blood mononuclear cell products have a larger population of helper T cells (TH, CD4+) than cytotoxic or suppressor T cells (TC, CD8 +). Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors produces a population of T cells consisting of a preponderance of TH cells about day 8-9 ago, while after about day 8-9, the population of T cells comprises an increased population of larger TC cells. Thus, depending on the purpose of the treatment, it may be advantageous to infuse the subject with a population of T cells predominantly comprising TH cells. Similarly, if an antigen-specific subpopulation of TC cells has been isolated, it may be beneficial to expand this subpopulation to a greater extent.

In addition, other phenotypic markers, in addition to CD4 and CD8 markers, also vary significantly, but most can vary reproducibly during the cell expansion process. This reproducibility thus makes it possible to tailor the ability of activated T cell products for specific purposes.

Once the CD33 CAR is constructed, various assays can be used to evaluate the activity of the molecule, such as, but not limited to, the ability to expand T cells following antigen stimulation, the ability to maintain T cell expansion in the absence of re-stimulation, and anti-cancer activity in suitable in vitro and animal models. Assays to evaluate the effects of CD33 CAR are described in further detail below.

Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers. See, e.g., Milone et al, Molecular Therapy 17(8):1453-1464 (2009). Very briefly, T cells expressing CAR (CD 4)⁺1:1 mixture of T cells and CD8+ T cells) was expanded in vitro for more than 10 days, followed by lysis and SDS-PAGE under reducing conditions. CARs containing the full-length TCR-zeta cytoplasmic domain and endogenous TCR-zeta chain were detected by western blot using antibodies against the TCR-zeta chain. The same T cell subpopulation was used for SDS-PAGE analysis under non-reducing conditions to allow evaluation of covalent dimer formation.

The in vitro expansion of CAR + T cells following antigen stimulation can be measured by flow cytometry. For example, CD4⁺The mixture of T cells and CD8+ T cells was stimulated with α CD3/α CD28aAPC and subsequently transduced with a lentiviral vector expressing GFP under the control of the promoter to be analyzed. Exemplary promoters include the CMV IE gene promoter, the EF-1 alpha promoter, the ubiquitin C promoter, or the phosphoglycerate kinase (PGK) promoter. Evaluation of CD4 by flow cytometry at day 6 of culture⁺And/or GFP fluorescence in CD8+ T cell subsets. See, e.g., Milone et al, Molecular Therapy 17(8):1453-1464 (2009). Alternatively, CD4 ⁺A mixture of T cells and CD8+ T cells was stimulated with α CD3/α CD28 coated magnetic beads on day 0 and transduced with CAR on day 1 using a bicistronic lentiviral vector, wherein the bicistronic lentiviral vector expresses CAR along with eGFP using a 2A ribosome skip sequence. After washing, the cultures were washed with CD19⁺K562 cells (K562-CD19), wild type K562 cells (K562 wild type) or K562 cells expressing hCD32 and 4-1BBL were restimulated in the presence of anti-CD 3 and anti-CD 28 antibodies (K562-BBL-3/28). Exogenous IL-2 was added to the cultures at 100IU/ml every 1 day. Counting GFP by flow cytometry Using a bead-based counting method⁺T cells. See, e.g., Milone et al, Molecular Therapy 17(8):1453-1464 (2009). Similar assays can be performed using anti-CD 123T cells (see, e.g., Gill et al, Blood 2014; 123:2343) or with anti-CD 33 CAR T cells.

CAR + T cell expansion maintained in the absence of restimulation can also be measured. See, e.g., Milone et al, Molecular Therapy 17(8):1453-1464 (2009). Briefly, after stimulation with α CD3/α CD28 coated magnetic beads on day 0 and transduction with the indicated CAR on day 1, mean T cell volume (fl) was measured on day 8 of culture using a Coulter size Analyzer III particle counter Nexcellometer Vision or Millipore Scepter.

Animal models can also be used to measure CART activity. For example, use of human CD 19-specific CARs in immunodeficient mice can be used⁺T cell treatment primary human pre-B ALL xenograft models. See, e.g., Milone et al, Molecular Therapy 17(8):1453-1464 (2009). Very briefly, after ALL was established, mice were randomly assigned to treatment groups. Co-injection of different numbers of alpha CD 19-zeta engineered T cells and alpha CD 19-BB-zeta engineered T cells at a 1:1 ratio into NOD-SCID-gamma carrying B-ALL^-/-In mice. The number of copies of α CD 19-zeta vector and α CD 19-BB-zeta vector in spleen DNA from mice was evaluated at various times after T cell injection. Animals were evaluated for leukemia at weekly intervals. Measurement of peripheral blood CD19 in mice injected with alpha CD 19-zeta CAR + T cells or mock-transduced T cells⁺B-ALL blast counts. Survival curves for each group were compared using the log rank test. In addition, NOD-SCID-gamma can also be analyzed^-/- Peripheral blood CD4 4 weeks after T cell injection in mice⁺And CD8⁺Absolute T cell counts. Mice were injected with leukemic cells and 3 weeks later with T cells engineered to express CAR via a bicistronic lentiviral vector encoding a CAR linked to eGFP. T cells were paired with 45-50% infused GFP by mixing with mock-transduced cells prior to injection ⁺T cells were normalized and confirmed by flow cytometry. Animals were evaluated for leukemia at 1 week intervals. Using log rank test, ratioCompare CAR⁺Survival curves for T cell groups. Similar experiments can be performed with CD33 CART.

Dose-dependent CAR treatment responses can be evaluated. See, e.g., Milone et al, Molecular Therapy 17(8):1453-1464 (2009). For example, peripheral blood is obtained 35-70 days after the establishment of leukemia in mice injected with CAR T cells, an equivalent number of mock-transduced T cells, or no T cells on day 21. Mice from each group were randomly bled to determine peripheral blood CD19⁺ALL blast cells were counted and subsequently killed on days 35 and 49. The remaining animals were evaluated on days 57 and 70. Similar experiments can be performed with CD33 CART.

Assessment of cell proliferation and cytokine production has been previously described, for example, in Milone et al, Molecular Therapy 17(8):1453-1464 (2009). Briefly, CAR-mediated proliferation was assessed in microtiter plates by mixing washed T cells with either K562 cells expressing CD19(K19) or K562 cells expressing CD32 and CD137 (KT32-BBL) in a final ratio of T cells to K562 of 2: 1. K562 cells were irradiated with gamma-radiation prior to use. anti-CD 3 (clone OKT3) monoclonal antibody and anti-CD 28 (clone 9.3) monoclonal antibody were added to the cultures with KT32-BBL cells acting as positive controls to stimulate T cell proliferation, as these signals support long-term ex vivo expansion of CD8+ T cells. CountBright was used as described by the manufacturer ^TMFluorescent beads (Invitrogen, Carlsbad, CA) and flow cytometry, the T cells in culture were counted. Identification of CARs by GFP expression using T cells⁺A T cell, wherein the T cell is engineered with a lentiviral vector expressing an eGFP-2A-linked CAR. For CAR that does not express GFP⁺T cells, detection of CAR with biotinylated recombinant CD33 protein and second Antibiotinoprotein-PE conjugate⁺T cells. CD4 on T cells was also simultaneously detected with specific monoclonal antibodies (BD Biosciences)⁺Expression and CD8⁺And (4) expressing. Cytokine measurements were performed on supernatants collected 24 hours after restimulation using either the human TH1/TH2 cytokine cytometric bead array kit (BD Biosciences, San Diego, Calif.) or using the Luminex30-plex kit (Invitrogen) according to the manufacturer's instructions. Using BDFortess flow cytometry evaluated fluorescence and analyzed the data according to the manufacturer's instructions. Similar experiments can be performed with CD33 CART.

Can be made of standard⁵¹The Cr-release assay assesses cytotoxicity. See, e.g., Milone et al, Molecular Therapy 17(8):1453-1464 (2009). Briefly, target cells (K562 lines and primary pre-B-ALL cells) were used at 37 deg.C⁵¹Cr (as NaCrO)₄New England Nuclear, Boston, MA) was loaded for 2 hours with frequent stirring, washed 2 times in complete RPMI and placed into microtiter plates. In the wells, effector T cells were mixed with target cells in a complete RPMI at different effector to target cell ratios (E: T). Additional wells containing either media only (spontaneous release, SR) or 1% triton-X100 detergent solution (complete release, TR) were also prepared. After 4 hours of incubation at 37 ℃, supernatants were harvested from each well. The released was then measured using a gamma particle counter (Packard Instrument co., Waltham, MA) ⁵¹And Cr. Each condition was repeated at least three times and the percent cleavage was calculated using the formula: percent cleavage ═ ER-SR)/(TR-SR), where ER represents per experimental condition⁵¹And average release of Cr.

Imaging techniques can be used to assess the specific transport and proliferation of CARs in tumor-bearing animal models. Such assays have been described, for example, in Barrett et al, Human Gene Therapy 22:1575-1586 (2011). Briefly, NOD/SCID/gammac pairs^–/–(NSG) mice were injected intravenously with Nalm-6 cells, 7 days later, 4 hours after electroporation with the CAR construct, and intravenously with T cells. T cells were stably transfected with a lentiviral construct expressing firefly luciferase and bioluminescence of mice was imaged. Alternatively, a single injection of CAR in the Nalm-6 xenograft model can be measured as follows⁺Therapeutic efficacy and specificity of T cells: NSG mice were injected with Nalm-6 transduced to stably express firefly luciferase, 7 days later, followed by a single tail vein injection of T cells electroporated with CD33 CAR. Animals were imaged at various time points post-injection. For example, day 5 (2 days before treatment) and day 8 (CAR) can be used⁺24 hours after PBL) generated photon dense for firefly luciferase-positive leukemia in representative mice A heat map. Other assays, including those described in the examples section herein and those known in the art, may also be used to evaluate the CD33 CAR constructs of the invention.

Alternatively or in combination with the methods disclosed herein, methods and compositions are disclosed that involve the use of one or more of the following CAR ligands: detecting and/or quantifying cells expressing the CAR (e.g., in vitro or in vivo (e.g., clinical monitoring)); immune cell expansion and/or activation; and/or CAR-specific selection. In an exemplary embodiment, the CAR ligand is an antibody that binds to a CAR molecule, e.g., an antibody that binds to an extracellular antigen-binding domain of the CAR (e.g., an antibody that binds to an antigen-binding domain, e.g., an anti-idiotypic antibody; or an antibody that binds to a constant region of an extracellular binding domain). In other embodiments, the CAR ligand is a CAR antigen molecule (e.g., a CAR antigen molecule as described herein).

In one aspect, a method for detecting and/or quantifying a CAR-expressing cell is disclosed. For example, the CAR ligand can be used to detect and/or quantify cells expressing the CAR in vitro or in vivo (e.g., clinically monitoring cells expressing the CAR in a patient or administering to a patient). The method comprises the following steps:

Providing a CAR ligand (optionally, a labeled CAR ligand, e.g., a CAR ligand comprising a tag, bead, radioactive or fluorescent label);

obtaining a CAR-expressing cell (e.g., obtaining a sample containing a CAR-expressing cell, such as a manufacturing sample or a clinical sample);

contacting the CAR-expressing cell with the CAR ligand under conditions in which binding occurs, thereby detecting the level (e.g., amount) of CAR-expressing cell present. Binding of CAR expressing cells to the CAR ligand can be detected using standard techniques such as FACS, ELISA, etc.

In another aspect, a method of expanding and/or activating a cell (e.g., an immune effector cell) is disclosed. The method comprises the following steps:

providing a CAR-expressing cell (e.g., a first CAR-expressing cell or a transiently CAR-expressing cell);

contacting the CAR-expressing cell with a CAR ligand (e.g., a CAR ligand as described herein) under conditions in which immune cell expansion and/or proliferation occurs, thereby generating an activated and/or expanded cell population.

In certain embodiments, the CAR ligand is present on (e.g., immobilized to or conjugated to) a substrate (e.g., a non-naturally occurring substrate). In some embodiments, the substrate is a non-cellular substrate. The non-cellular substrate can be a solid support selected from, for example, a plate (e.g., a microtiter plate), a membrane (e.g., a nitrocellulose membrane), a matrix, a chip, or a bead. In embodiments, the CAR ligand is present in the substrate (e.g., on the surface of the substrate). The CAR ligand may be covalently or non-covalently immobilized, linked, or conjugated (e.g., crosslinked) to a substrate. In one embodiment, the CAR ligand is attached (e.g., covalently attached) to the bead. In the foregoing embodiments, the population of immune cells may be expanded in vitro or ex vivo. Such methods can further comprise culturing the population of immune cells in the presence of the ligand of the CAR molecule, e.g., using any of the methods described herein.

In other embodiments, the method of expanding and/or activating cells further comprises adding a second stimulatory molecule, e.g., CD 28. For example, the CAR ligand and the second stimulatory molecule can be immobilized to a substrate, e.g., one or more beads, thus providing increased cell expansion and/or activation.

In yet another aspect, a method for selecting or enriching for a cell expressing a CAR is provided. The method comprises contacting a cell expressing a CAR with a CAR ligand as described herein; and selecting the cell based on the binding of the CAR ligand.

In yet other embodiments, a method for depleting, reducing, and/or killing a CAR-expressing cell is provided. The method comprises contacting a cell expressing a CAR with a CAR ligand as described herein; and targeting the cell based on the binding of the CAR ligand, thereby reducing the number of and/or killing the CAR-expressing cell. In one embodiment, the CAR ligand is conjugated to a toxic substance (e.g., a toxin or a cytoablative drug). In another embodiment, the anti-idiotype antibody may cause effector cell activity, e.g., ADCC or ADC activity.

Exemplary anti-CAR antibodies that can be used in the methods disclosed herein are described, for example, in WO2014/190273 and by Jen et al, "Chimeric Antibody Receptor (CAR) -Specific Monoclonal Antibody to Detect CD19-Specific T cells in Clinical Trials", PLOS March 20138: 3 e57838, the contents of which are incorporated by reference. In one embodiment, the anti-idiotype antibody molecule recognizes an anti-CD 19 antibody molecule, e.g., an anti-CD 19 scFv. For example, an anti-idiotype antibody molecule can compete for binding to CD19-specific CAR mAb clone No. 136.20.1 described in Jena et al, PLOS March 20138: 3 e 57838; can have the same CDRs as CD19-specific CAR mAb clone number 136.20.1 (e.g., using Kabat definitions, Chothia definitions, or a combination of Kabat and Chothia definitions, one or more (e.g., all) of VH CDR1, VH CDR2, CHCDR3, VL CDR1, VL CDR2, and VL CDR 3)); may have one or more (e.g., 2) variable regions as CD19-specific CAR mAb clone No. 136.20.1, or may comprise CD19-specific CAR mAb clone No. 136.20.1. In some embodiments, the anti-idiotype antibody is prepared according to one of the methods described in Jena et al. In another embodiment, the anti-idiotype antibody molecule is an anti-idiotype antibody molecule as described in WO 2014/190273. In some embodiments, the anti-idiotype antibody molecule has the same CDRs (e.g., one or more (e.g., all) of VH CDR1, VH CDR2, CHCDR3, VL CDR1, VL CDR2, and VL CDR 3) as the antibody molecule of WO2014/190273 such as 136.20.1; one or more (e.g., 2) variable regions of the antibody molecule of WO2014/190273 may be present, or the antibody molecule of WO2014/190273 such as 136.20.1 may be comprised. In other embodiments, the anti-CAR antibody binds to a constant region of the extracellular binding domain of the CAR molecule (e.g., as described in WO 2014/190273). In some embodiments, the anti-CAR antibody binds to a constant region of the extracellular binding domain of the CAR molecule (e.g., a heavy chain constant region (e.g., a CH2-CH3 hinge region) or a light chain constant region). For example, in some embodiments, the anti-CAR antibody competes for binding to the 2D3 monoclonal antibody described in WO2014/190273, has the same CDRs as 2D3 (e.g., one or more (e.g., all) of VH CDR1, VH CDR2, CHCDR3, VL CDR1, VL CDR2, and VL CDR 3), or has one or more (e.g., 2) variable regions of 2D3, or comprises 2D3 as described in WO 2014/190273.

In some aspects and embodiments, the compositions and methods herein are optimized for specific T cell subpopulations, for example, as described in U.S. serial No. 62/031,699 filed on 31/7/2014, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the T cell is a different type (e.g., expresses a different type of the same construct (e.g., CD 8) than the control T cell⁺Or CD4⁺) T cells) that exhibit enhanced persistence.

In some embodiments, CD4⁺The T cell comprises a CAR described herein comprising a suitable CD4⁺An intracellular signaling domain, e.g., ICOS domain, of a T cell (e.g., optimized for it, e.g., resulting in enhanced persistence therein). In some embodiments, CD8⁺The T cell comprises a CAR described herein comprising a suitable CD8⁺An intracellular signaling domain of a T cell (e.g., optimized for it, e.g., resulting in its enhanced persistence), e.g., a 4-1BB domain, a CD28 domain, or another costimulatory domain other than an ICOS domain. In some embodiments, the CAR described herein comprises an antigen binding domain described herein, e.g., a CAR comprising an antigen binding domain targeting CD33, e.g., a CAR of table 2 or a CAR having the amino acid sequence of SEQ ID NO:140, or an antigen binding domain comprising the amino acid sequence of SEQ ID NO: 147.

In one aspect, described herein is a method of treating a subject (e.g., a subject having cancer). The method comprises administering to the subject an effective amount of:

1) CD4 comprising CAR⁺T Cells (CAR)^CD4+)

The CAR comprises:

an antigen binding domain, e.g., an antigen binding domain described herein, e.g., an antigen binding domain targeting CD33, e.g., an antigen binding domain of table 2 or table 9, or an antigen binding domain comprising the amino acid sequence of SEQ ID NO:140 or 147;

a transmembrane domain; and

an intracellular signaling domain, e.g., a first costimulatory domain, e.g., an ICOS domain; and

2) CD8 comprising CAR⁺T Cells (CAR)^CD8+) The CAR comprises:

a transmembrane domain; and

an intracellular signaling domain, e.g., a second costimulatory domain, e.g., a 4-1BB domain, a CD28 domain, or another costimulatory domain other than an ICOS domain;

Wherein CAR^CD4+And CAR^CD8+Are different from each other.

Optionally, the method further comprises administering:

3) second CD8+ T cell comprising CAR (second CAR)^CD8+) The CAR comprises:

an antigen binding domain, e.g., an antigen binding domain described herein, e.g., an antigen binding domain that specifically binds to CD33, e.g., an antigen binding domain of table 2 or table 9, or an antigen binding domain comprising the amino acid sequence of SEQ ID NOs 140 or 147;

a transmembrane domain; and

an intracellular signaling domain, wherein the second CAR^CD8+Is comprised in CAR^CD8+An intracellular signaling domain not present, e.g., a costimulatory signaling domain, and optionally, does not comprise an ICOS signaling domain.

Therapeutic applications

CD33 related diseases and/or disorders

The invention provides, inter alia, methods for treating diseases associated with expression of CD33 or conditions associated with cells expressing CD33, including, for example, proliferative diseases such as cancer or malignancies or precancerous conditions such as myelodysplasia, myelodysplastic syndrome, or pre-leukemia; or a non-cancer related indication associated with cells expressing CD33, in one aspect, the cancer associated with CD33 is a hematologic cancer. In one aspect, hematologic cancers include, but are not limited to, AML, myelodysplastic syndrome, ALL, chronic myeloid leukemia, blast cell plasma-like dendritic cell tumors, myeloproliferative tumors, and the like. In addition, other diseases associated with expression of CD33 include, for example, but are not limited to, atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases associated with expression of CD 33. Non-cancer related indications associated with expression of CD33 may also be included.

In one aspect, the invention provides methods for treating diseases associated with expression of CD 33. In one aspect, the invention provides methods for treating diseases in which a portion of the tumors are CD33 negative and a portion of the tumors are CD33 positive. For example, the CARs of the invention can be used to treat a subject that has been treated for a disease associated with elevated CD33 expression, wherein a subject that has been treated for elevated CD33 levels exhibits a disease associated with elevated CD33 levels. In embodiments, the CARs of the invention are useful for treating a subject that has been treated for a disease associated with expression of CD33, wherein a subject that has received a treatment associated with expression of CD33 displays a disease associated with expression of CD 33.

In one aspect, the invention relates to a vector comprising a CD33 CAR operably linked to a promoter for expression in a mammalian immune effector cell (e.g., a T cell or NK cell). In one aspect, the invention provides a recombinant immune effector cell (e.g., a T cell or NK cell) that expresses a CD33 CAR for use in treating a CD 33-expressing tumor, wherein the recombinant immune effector cell (e.g., a T cell or NK cell) that expresses a CD33 CAR is referred to as a CD33 CAR-expressing cell (e.g., a CD33 CART or a CD33 CAR-expressing NK cell). In one aspect, a CAR-expressing cell of the invention (e.g., a CD33 CART or a CD33 CAR-expressing NK cell) is capable of contacting a tumor cell with at least one CD33 CAR of the invention expressed on its surface such that the CAR-expressing cell (e.g., a CD33 CART or a CD33 CAR-expressing NK cell) targets the tumor cell and inhibits growth of the tumor.

In one aspect, the invention relates to a method of inhibiting growth of a tumor cell expressing CD33, the method comprising contacting the tumor cell with a CD33 CAR-expressing cell of the invention (e.g., a CD33 CART or a CD33 CAR-expressing NK cell) such that the CAR-expressing cell (e.g., a CD33 CART or a CD33 CAR-expressing NK cell) activates and targets the cancer cell in response to an antigen, wherein growth of the tumor is inhibited.

In one aspect, the invention relates to a method of treating cancer in a subject. The method comprises so administering to the subject a cell expressing a CD33 CAR (e.g., a CD33 CART or a NK cell expressing a CD33 CAR), thereby treating the cancer in the subject. An example of a cancer treatable by a CD33 CAR-expressing cell of the invention (e.g., a CD33 CART or a CD33 CAR-expressing NK cell) is a cancer associated with expression of CD 33. Examples of cancers treatable by the CD33 CAR expressing cells of the invention (e.g., CD33 CART or CD33 CAR expressing NK cells) include, but are not limited to, AML, myelodysplastic syndrome, chronic myeloid leukemia and other myeloproliferative tumors, or blast cell plasma-like dendritic cell tumors, and the like.

The invention includes a type of cell therapy in which immune effector cells (e.g., T cells or NK cells) are genetically modified to express a Chimeric Antigen Receptor (CAR) and cells expressing the CAR (e.g., CD33 CART or NK cells expressing CD33 CAR) are infused to a recipient in need thereof. The infused cells are capable of killing tumor cells in the recipient. Unlike antibody therapies, CAR-modified cells (e.g., T cells or NK cells) are capable of replication in vivo, resulting in long-term persistence that can lead to persistent tumor control. In various aspects, following administration of cells (e.g., T cells or NK cells) to a patient, the cells (e.g., T cells or NK cells) or progeny thereof administered to the patient persist in the patient for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, or 5 years.

The invention also includes a type of cell therapy in which immune effector cells (e.g., T cells or NK cells) are modified (by in vitro transcribed RNA) to transiently express a Chimeric Antigen Receptor (CAR) and the immune effector cells (e.g., T cells or NK cells) are infused to a recipient in need thereof. The infused cells are capable of killing tumor cells in the recipient. Thus, in various aspects, following administration of immune effector cells (e.g., T cells or NK cells) to a patient, the immune effector cells (e.g., T cells or NK cells) administered to the patient are present for less than 1 month, e.g., 3 weeks, 2 weeks, 1 week.

Without wishing to be bound by any particular theory, the anti-tumor immune response elicited by the CAR-modified immune effector cells (e.g., T cells or NK cells) may be an active or passive immune response, or alternatively may be due to a direct vs. In one aspect, CAR-transduced immune effector cells (e.g., T cells or NK cells) exhibit specific pro-inflammatory cytokine secretion and potent cytolytic activity in response to CD 33-expressing human cancer cells, resist soluble CD33 inhibition, mediate bystander killing and mediate established human tumor regression. For example, tumor cells with fewer antigens within the internal heterogeneous field of view of a tumor expressing CD33 may be susceptible to indirect destruction by CD 33-redirected immune effector cells (e.g., T cells or NK cells) that have previously reacted against adjacent antigen-positive cancer cells.

In one aspect, the fully human CAR-modified immune effector cell (e.g., T cell or NK cell) of the invention can be a vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one aspect, the mammal is a human.

For ex vivo immunization, at least one of the following procedures is performed in vitro prior to administration of the cells to the mammal: i) expanding the cell, ii) introducing a nucleic acid encoding a CAR into the cell, or iii) cryopreserving the cell.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with the CAR-expressing vectors disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the CAR-modified cells may be autologous with respect to the recipient. Alternatively, the cells may be allogeneic, syngeneic, or xenogeneic with respect to the recipient.

Methods of ex vivo expansion of hematopoietic stem and progenitor cells are described in U.S. Pat. No. 5,199,942, incorporated herein by reference, and may be applied to the cells of the present invention. Other suitable methods are known in the art, and thus, the present invention is not limited to any particular method of expanding cells ex vivo. Briefly, ex vivo culture and expansion of T cells includes: (1) harvesting CD34+ hematopoietic stem cells and progenitor cells from a mammal from a peripheral blood harvest or bone marrow explant; and (2) ex vivo expansion of such cells. In addition to the cell growth factors described in U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligands may also be used in the culture and expansion of cells.

In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

In general, cell activation and expansion as described herein can be used to treat and prevent diseases that arise in immunocompromised individuals. In particular, the CAR-modified immune effector cells (e.g., T cells or NK cells) of the invention are useful for treating diseases, disorders, and conditions associated with expression of CD 33. In certain aspects, the cells of the invention are used to treat patients at risk of developing diseases, disorders, and conditions associated with expression of CD 33. Accordingly, the present invention provides methods for treating or preventing diseases, disorders, and conditions associated with expression of CD33, comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified immune effector cell (e.g., a T cell or NK cell) of the invention.

In one aspect, the CAR-expressing cells of the invention (e.g., CART cells or CAR-expressing NK cells) can be used to treat a proliferative disease such as a cancer or malignancy or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or pre-leukemia. In one aspect, the cancer associated with expression of CD33 is a hematologic cancer, a pre-leukemia, a hyperproliferative disease, a hyperplasia or a dysplasia characterized by abnormal cell growth.

In one aspect, the CAR-expressing cells of the invention (e.g., CART cells or CAR-expressing NK cells) are used to treat cancer, wherein the cancer is a hematologic cancer. Hematologic cancer diseases are many types of cancer that affect the blood, myeloid, and lymphatic systems, such as leukemia and malignant lymphoproliferative disorders.

In one aspect, the compositions and CAR-expressing cells (e.g., CART cells or CAR-expressing NK cells) of the invention are particularly useful for treating myeloid leukemia, AML and its subtypes, Chronic Myeloid Leukemia (CML), and myelodysplastic syndrome (MDS).

Leukemias can be divided into acute leukemias and chronic leukemias. Acute leukemias can be further classified into Acute Myeloid Leukemia (AML) and Acute Lymphoid Leukemia (ALL). Chronic leukemias include Chronic Myelogenous Leukemia (CML) and Chronic Lymphoid Leukemia (CLL). Other related diseases include myelodysplastic syndrome (MDS, formerly known as "preleukemia"), which is a diverse collection of hematological diseases that are unified by the ineffective production (or dysplasia) of myeloid blood cells, and the risk of conversion to AML.

Lymphomas are a group of blood cell tumors that develop from lymphocytes. Exemplary lymphomas include non-hodgkin lymphoma and hodgkin lymphoma.

In AML, malignant and uncontrolled proliferation of abnormally differentiated long-lived myeloid progenitors results in high cycle numbers of immature blood forms and malignant cells replacing normal bone marrow. Symptoms include fatigue, pale complexion, easy bruising and bleeding, fever and infection; symptoms of leukemic infiltration are present in only about 5% of patients (often manifested as skin). The detection of peripheral blood smears and bone marrow is of diagnostic significance. Existing treatments include induction chemotherapy to achieve remission and post-remission chemotherapy (with or without stem cell transplantation) to avoid relapse.

AML has numerous subtypes that are distinguished from each other by morphology, immunophenotype, and cytochemistry. Based on the predominant cell type, five types are described, including myeloid, myeloid-monocytic, erythroid, and megakaryocytic.

The induction of remission is 50% to 85%. Long-term disease-free survival has been reported to occur in 20% to 40% of patients and to increase to 40% to 50% in younger patients treated with stem cell transplantation.

Prognostic factors help determine treatment regimens and strengths; patients with strong poor prognostic characteristics are often given a more aggressive form of therapy because the potential benefits are believed to justify increased toxicity of the treatment. The most important prognostic factors are the leukemia cell karyotype; advantageous karyotypes include t (15; 17), t (8; 21), and inv16(p 13; q 22). Adverse factors include increasing age, previous myelodysplastic period, secondary leukemia, high WBC count and absence of Auer bodies (Auer rods).

Initial therapy attempts to induce remission and is the most different from ALL because AML responds to fewer drugs. The basal induction treatment regimen comprises continuous intravenous infusion or bolus of cytarabine for 5 to 7 days; over this time period zorubicin or idarubicin was administered intravenously for 3 days. Some regimens include 6-thioguanine, etoposide, vincristine and prednisone, but their contributions are not clear. Treatment often results in significant bone marrow suppression, with infection or bleeding; there is a significant delay time before bone marrow recovery. During this time careful preventive and supportive care is of paramount importance.

Chronic myelogenous (or myeloid) leukemia (CML) is also known as chronic myeloid leukemia and is characterized by a form of leukemia. Common CML treatment regimens include the Bcr-Abl tyrosine kinase inhibitor imatinib

Dasatinib and nilotinib. Bcr-Abl tyrosine kinase inhibitors are particularly useful in CML patients with Philadelphia chromosomal translocations.

Myelodysplastic syndrome (MDS) is a hematological disease characterized by disorganized and ineffective hematopoiesis or blood production. Thus, the number and quality of blood-forming cells irreversibly decrease. Some subjects with MDS may develop severe anemia, while others are asymptomatic. The classification scheme for MDS is known in the art with criteria that specify the ratio or frequency of particular blood cell types (e.g., myeloblasts, monocytes, and erythrocyte precursors). MDS includes refractory anemia, refractory anemia with ringed sideroblasts (ring sideroblasts), refractory anemia with excess blasts transformed, Chronic Myelomonocytic Leukemia (CML).

Treatment of MDS varies with the severity of the symptoms. Aggressive forms of treatment for patients presenting with severe symptoms include bone marrow transplantation and supportive therapy with blood product support (e.g., transfusion) and hematopoietic growth factors (e.g., erythropoietin). Other drugs are frequently used to treat MDS: 5-azacytidine, decitabine, and lenalidomide. In some cases, the iron chelator deferoxamine may also be administered

And deferasirox

In another embodiment, the CAR-expressing cells of the invention (e.g., CART cells or CAR-expressing NK cells) are used to treat cancer or leukemia with leukemic stem cells. For example, the leukemia stem cell is CD34⁺/CD38^-Leukemia cells.

The invention provides, inter alia, compositions and methods for treating cancer. In one aspect, the cancer is a hematologic cancer, including, but not limited to, leukemias (e.g., acute myelocytic leukemia, chronic myelogenous leukemia, acute lymphoid leukemia, chronic lymphoid leukemia, and myelodysplastic syndrome) and malignant lymphoproliferative disorders, including lymphomas (e.g., multiple myeloma, non-hodgkin's lymphoma, Burkitt's lymphoma, and small-and large-cell-follicular lymphoma).

In one aspect, the CAR-expressing cells of the invention (e.g., CART cells or CAR-expressing NK cells) can be used to treat other cancers and malignancies, such as, but not limited to, for example, acute leukemias, including, but not limited to, e.g., B-cell acute lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"), Acute Lymphoid Leukemia (ALL); one or more chronic leukemias, including, but not limited to, for example, Chronic Myelogenous Leukemia (CML), Chronic Lymphocytic Leukemia (CLL); additional hematologic cancers or hematologic diseases, including, but not limited to, for example, B cell prolymphocytic leukemia, blastic plasma cell-like dendritic cell tumor, Burkitt lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative disease, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-hodgkin lymphoma, plasmablast lymphoma, plasma cell-like dendritic cell tumor, Waldenstrom's macroglobulinemia, and "preleukemia" as a collection of diverse hematologic diseases that are linked by ineffective production (or dysplasia) of myeloid blood cells, and the like. The CAR-modified immune effector cells (e.g., T cells or NK cells) of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.

The present invention also provides a method for inhibiting proliferation of a population of cells expressing CD33 or reducing a population of cells expressing CD33, the method comprising: contacting a population of cells comprising cells expressing CD33 with a CD33 CAR-expressing cell of the invention (e.g., a CD33 CAR T cell or a CD33 CAR-expressing NK cell) that binds to a CD 33-expressing cell. In a particular aspect, the invention provides a method for inhibiting the proliferation of a population of cancer cells expressing CD33 or reducing a population of cancer cells expressing CD33, the method comprising: contacting a population of cells comprising CD 33-expressing cancer cells with a CD33 CAR-expressing cell of the invention (e.g., a CD33 CAR T cell or a CD33 CAR-expressing NK cell) that binds to a CD 33-expressing cell. In one aspect, the present invention provides a method for inhibiting the proliferation of a population of cancer cells expressing CD33 or reducing a population of cancer cells expressing CD33, the method comprising: contacting a population of cells comprising CD 33-expressing cancer cells with a CD33 CAR-expressing cell of the invention (e.g., a CD33 CAR T cell or a CD33 CAR-expressing NK cell) that binds to a CD 33-expressing cell. In certain aspects, in a subject having myeloid leukemia or another cancer associated with cells expressing CD33, or an animal model of the foregoing, the CD33 CAR-expressing cells of the invention (e.g., CD33 CART cells or CD33 CAR-expressing NK cells reduce the amount, number, or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% relative to a negative control.

The present invention also provides methods for preventing, treating and/or managing a disease associated with cells expressing CD33 (e.g., a hematologic cancer or atypical cancer that expresses CD 33), the method comprising: administering to a subject in need thereof a CD33 CAR-expressing cell of the invention (e.g., a CD33 CART cell or a NK cell expressing a CD33 CAR) that binds to a CD 33-expressing cell. In one aspect, the subject is a human. Non-limiting examples of conditions associated with cells expressing CD33 include autoimmune diseases (such as lupus), inflammatory diseases (such as allergy and asthma), and cancer (such as hematologic or atypical cancers that express CD 33).

The present invention also provides a method for preventing, treating and/or managing a disease associated with cells expressing CD33, the method comprising: administering to a subject in need thereof a CD33 CAR-expressing cell of the invention (e.g., a CD33 CART cell or a NK cell expressing a CD33 CAR) that binds to a CD 33-expressing cell. In one aspect, the subject is a human.

The present invention provides a method for preventing recurrence of cancer associated with cells expressing CD33, the method comprising: administering to a subject in need thereof a CD33 CAR-expressing cell of the invention (e.g., a CD33 CART cell or a NK cell expressing a CD33 CAR) that binds to a CD 33-expressing cell. In one aspect, the method comprises administering to a subject in need thereof an effective amount of an anti-CD 33 expressing cell described herein (e.g., a CD33 CART cell or a CD33 CAR expressing NK cell) that binds to a CD33 expressing cell in combination with an effective amount of another treatment.

Combination therapy

The CAR-expressing cells described herein can be used in combination with other known drugs and therapies. As used herein, "administering in combination" means delivering two or more different treatments to a subject during the subject's suffering from a disorder, e.g., delivering two or more treatments after the subject has been diagnosed with the disorder and before the disorder has cured or eliminated or the treatment has been stopped for other reasons. In some embodiments, delivery of one therapy is still ongoing when delivery of a second therapy is initiated, such that there is overlap with respect to administration. This is sometimes referred to herein as "simultaneous" or "synchronous delivery". In other embodiments, the delivery of one treatment ends before the delivery of the second treatment begins. In some embodiments of either case, the treatment is more effective because of the combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is observed when the second treatment is less, or the second treatment reduces symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a similar situation was observed with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than would be observed if one treatment were delivered in the absence of the other treatment. The effects of the two therapeutic agents may be partially additive, fully additive, or more than additive. The delivery may be such that when the second therapeutic agent is delivered, the effect of the first therapeutic agent is still detectable.

The CAR-expressing cells described herein and the at least one additional therapeutic agent can be administered simultaneously or sequentially in the same composition or in separate compositions. For sequential administration, the CAR-expressing cells described herein can be administered first and the additional drug can be administered next, or the order of administration can be reversed.

CAR therapy and/or other therapeutic agents, procedures or modes may be administered during active disease time or during remission or less active disease time. CAR therapy can be administered prior to other treatment, concurrently with the treatment, after the treatment, or during remission of the disease.

When administered in combination, the CAR therapy and the additional drug (e.g., the second or third drug) or both can be administered in an amount or dose that is higher, lower, or equal to the amount or dose of each drug used alone (e.g., as a monotherapy). In certain embodiments, the CAR therapy, additional agent (e.g., second or third agent), or all of the former is administered at a lower amount or dose (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dose of each agent used alone (e.g., as monotherapy). In other embodiments, the amount or dose of CAR therapy, additional drug (e.g., second or third drug), or all of the former that produces the desired effect (e.g., treating cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dose of each drug used alone (e.g., as monotherapy) that is required to achieve the same therapeutic effect.

In other aspects, the CAR-expressing cells described herein can be used in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporine, azathioprine, methotrexate, mycophenolate salts and FK506, antibodies, or other immune scavengers such as CAMPATH, anti-CD 3 antibodies or other antibody therapeutics, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and radiation, peptide vaccines (such as those described in Izumoto et al, 2008J Neurosurg 108: 963-971) in a therapeutic regimen.

In certain instances, the compounds of the present invention are combined with other therapeutic agents such as other anticancer agents, antiallergic agents, anti-nausea agents (or anti-emetics), analgesics, cell protective agents, and combinations thereof.

In one embodiment, the CAR-expressing cells described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab (tositumomab), bernetuzumab), antimetabolites (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-induced TNFR-related protein (GITR) agonists; proteasome inhibitors (e.g., aclacinomycin a, gliotoxin, or bortezomib), immunomodulators such as thalidomide or thalidomide derivatives (e.g., lenalidomide).

Typical chemotherapeutic agents contemplated for use in combination therapy include busulfan

Busulfan injection

Claritabine

Cyclophosphamide (b)

Or

) Cytarabine and pyrimidine arabinoside

Cytarabine liposome injection

Zorubicin hydrochloride

Liposome injection of zorubicin citrate

Dexamethasone and doxorubicin hydrochloride

Etoposide

Fludarabine phosphate

Hydroxyurea

Idarubicin (Idarubicin)

Mitoxantrone (mitoxantrone), Gemtuzumab ozolomide (Gemtuzumab ozogamicin)

In embodiments, a general chemotherapeutic contemplated for use in combination therapy includes anastrozole

Bicalutamide

Bleomycin sulfate

Busulfan medicine

Busulfan injection

Capecitabine

N4-pentyloxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin

Carmustine

Chlorambucil

Cis-platinum

Claritabine

Cyclophosphamide (b)

Or

) Cytarabine and pyrimidine arabinoside

Cytarabine liposome injection

Dacarbazine

Dactinomycin (actinomycin D, Cosmegan), zorubicin hydrochloride

Liposome injection of zorubicin citrateAgent for treating cancer

Dexamethasone and docetaxel

Doxorubicin hydrochloride

Etoposide

Fludarabine phosphate

5-Fluorouracil

Flutamide

Tizacitabine (tezacitibine), gemcitabine (diflupredeoxycidine), hydroxyurea

Idarubicin (Idarubicin)

Isocyclophosphamide (ACS)

Irinotecan

L-asparaginase

Formyl tetrahydrofolic acid calcium, melphalan

6-mercaptopurine

Methotrexate (MTX)

Mitoxantrone (mitoxantrone), milotarg, paclitaxel

Phoenix (yttrium 90/MX-DTPA), pentostatin, polifeprosan 20 along with carmustine implants

Tamoxifen citrate

Teniposide

6-thioguanine, thiotepa and tirapazamine

Topotecan hydrochloride for injection

Vinblastine

Vincristine

And vinorelbine

Particularly interesting anticancer agents in combination with the compounds of the present invention include: anthracyclines; an alkylating agent; an antimetabolite; drugs that inhibit calcineurin, a calcineurin, or p70S6 kinase FK506), or inhibit p70S6 kinase; an mTOR inhibitor; an immunomodulator; anthracyclines; vinca alkaloids; a proteasome inhibitor; a GITR agonist; protein tyrosine phosphatase inhibitors; inhibitors of CDK kinases; a BTK inhibitor; an MKN kinase inhibitor; inhibitors of DGK kinase; or an oncolytic virus.

Exemplary antimetabolites include, but are not limited to, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors): methotrexate (MTX)

5-Fluorouracil

Floxuridine

Cytarabine (b)

Tarabine PFS), 6-mercaptopurine

) 6-thioguanine (thioguanine)

) Fludarabine phosphate

Pentostatin

Pemetrexed

Raltitrexed

Cladribine

Clofarabine

Azacitidine

Decitabine and gemcitabine

Preferred antimetabolites include cytarabine, clofarabine and fludarabine.

Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes): uracil mustard (Aminouracil)

Uracil mustard,

)、chlormethine

Cyclophosphamide (b)

Revimmune^TM) Ifosfamide (I) and (II)

Melphalan

Chlorambucil

Pipobroman

Triethylenemelamine

Triethylene thiophosphoramide and temozolomide

Thiotepa

Busulfan medicine

Carmustine

Luolimus extract

Chain zotard

And dacarbazine

Additional exemplary alkylating agents include, but are not limited to, oxaliplatin

Temozolomide (A)

And

) (ii) a Dactinomycin (also known as actinomycin-D, dactinomycin),

) (ii) a Melphalan (also known as L-PAM, L-escoline and melphalan,

) (ii) a Altretamine (Altretamine) (also known as Hexamethylmelamine (HMM)),

) (ii) a Carmustine

Bendamustine

Busulfan (Busulfan)

And

) (ii) a Carboplatin

Sirolimus (also known as CCNU,

) (ii) a Cisplatin (also known as CDDP,

And

) (ii) a Chlorambucil

Cyclophosphamide (b)

And

) (ii) a Dacarbazine (also known as DTIC, DIC and Imidazamide),

) (ii) a Altretamine (Altretamine) (also known as Hexamethylmelamine (HMM)),

) (ii) a Isocyclophosphamide (ACS)

Prednumustine; procarbazine

Mechlorethamine (also known as mechlorethamine, chlorazine and chlorambucil hydrochloride),

) (ii) a Chain zotard

Thiotepa (also known as thiophosphamide, TESPA and TSPA),

) (ii) a Cyclophosphamide

And bendamustine HCl

In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with fludarabine, cyclophosphamide, and/or rituximab. In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with fludarabine, cyclophosphamide, and rituximab (FCR). In embodiments, the subject has CLL. For example, the subject has a deletion in the short arm of chromosome 17 (del (17p) (e.g., in leukemia cells). in other examples, the subject does not have del (17 p). in embodiments, the subject is comprised of an immunoglobulinProtein heavy chain variable region (IgV)_H) Leukemia cells comprising a mutation in the gene. In other embodiments, the subject does not comprise an immunoglobulin heavy chain variable region (IgV) _H) Leukemia cells comprising a mutation in the gene. In embodiments, fludarabine is administered at the following doses: about 10-50mg/m²(e.g., about 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50mg/m²) For example, intravenous administration. In embodiments, cyclophosphamide is administered at the following dose: about 200-300mg/m²(e.g., about 200-225, 225-250, 250-275, or 275-300mg/m²) For example, intravenous administration. In embodiments, rituximab is administered at the following dose: about 400-600mg/m²(e.g., 400-450, 450-500, 500-550, or 550-600mg/m²) For example, intravenous administration.

In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with bendamustine and rituximab. In embodiments, the subject has CLL. For example, the subject has a deletion in the short arm of chromosome 17 (del (17p) (e.g., in leukemia cells). in other examples, the subject does not del (17 p). in embodiments, the subject comprises an immunoglobulin heavy chain variable region (IgV)_H) Leukemia cells comprising a mutation in the gene. In other embodiments, the subject does not comprise an immunoglobulin heavy chain variable region (IgV) _H) Leukemia cells comprising a mutation in the gene. In embodiments, bendamustine is administered at the following doses: about 70-110mg/m²(e.g., 70-80, 80-90, 90-100, or 100-110mg/m²) For example, intravenous administration. In embodiments, rituximab is administered at the following dose: about 400-600mg/m²(e.g., 400-450, 450-500, 500-550, or 550-600mg/m²) For example, intravenous administration.

In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and/or a corticosteroid (e.g., prednisone). In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). In embodiments, the subject has diffuse large B-cell lymphoma (DLBCL). In embodiments, the subject has a non-large, limited stage DLBCL (e.g., including a tumor with a size/diameter of less than 7 cm). In embodiments, the subject is treated with radiation in combination with R-CHOP. For example, the subject is administered R-CHOP (e.g., 1-6 cycles, e.g., 1, 2, 3, 4, 5, or 6 cycles of R-CHOP) followed by irradiation. In some cases, the R-CHOP is administered to the subject after irradiation (e.g., 1-6 cycles, e.g., 1, 2, 3, 4, 5, or 6 cycles of R-CHOP).

In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and/or rituximab. In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (EPOCH-R). In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with an adjusted dose of EPOCH-R (DA-EPOCH-R). In embodiments, the subject has a B-cell lymphoma, e.g., a Myc-rearranged aggressive B-cell lymphoma.

In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with rituximab and/or lenalidomide. Lenalidomide ((RS) -3- (4-amino-1-oxo-1, 3-dihydro-2H-isoindol-2-yl) piperidine-2, 6-dione) is an immunomodulator. In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with rituximab and lenalidomide. In embodiments, the subject has Follicular Lymphoma (FL) or Mantle Cell Lymphoma (MCL). In embodiments, the subject has FL and has not been previously treated with cancer therapy. In embodiments, lenalidomide is administered in the following doses: about 10-20mg (e.g., 10-15 or 15-20mg), e.g., once daily. In embodiments, rituximab is administered at the following dose: about 350-550mg/m ²(e.g., 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500)mg/m²) For example, intravenous administration.

Exemplary mTOR inhibitors include, for example, temsirolimus; desmopolimus (formally known as deferolimus, (1R,2R,4S) -4- [ (2R) -2[ (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R) -1, 18-dihydroxy-19, 30-dimethoxy-15, 17,21,23,29, 35-hexamethyl-2, 3,10,14, 20-pentaoxo-11, 36-dioxa-4-azatricyclo [30.3.1.0 [ -30.3.1.0 ]^4,9]Thirty-seven carbon-16, 24,26, 28-tetraen-12-yl]Propyl radical]2-Methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described in PCT publication No. WO 03/064383); everolimus (A)

Or RAD 001); rapamycin (AY22989,

) (ii) a Simapimod (CAS 164301-51-3); emirolimus, (5- {2, 4-bis [ (3S) -3-methylmorpholin-4-yl)]Pyrido [2,3-d]Pyrimidin-7-yl } -2-methoxyphenyl) methanol (AZD 8055); 2-amino-8- [ trans-4- (2-hydroxyethoxy) cyclohexyl]-6- (6-methoxy-3-pyridyl) -4-methyl-pyrido [2,3-d]Pyrimidin-7 (8H) -one (PF04691502, CAS 1013101-36-4); and N²- [1, 4-dioxo- [ [4- (4-oxo-8-phenyl-4H-1-benzopyran-2-yl) morpholinium-4-yl]Methoxy radical]Butyl radical]-L-arginylglycyl-L-alpha-aspartylL-serine (SEQ ID NO:378), inner salts (SF1126, CAS936487-67-1) and XL 765.

Exemplary immunomodulators include, for example, afutazumab (orimab)

Available)); PEGylated filgrastim

Lenalidomide (CC-5013, Relimide)

) (ii) a Thalidomide

actimid (CC 4047); and IRX-2 (a mixture of human cytokines including interleukin 1, interleukin 2, and interferon γ, CAS951209-71-5, available from IRX Therapeutics).

Exemplary anthracyclines include, for example, doxorubicin (doxorubicin and doxorubicin)

) (ii) a Bleomycin

Zorubicin (zorubicin hydrochloride, daunorubicin, and rubicin hydrochloride),

) (ii) a Liposomal zorubicin (liposomal zorubicin citrate,

) (ii) a Mitoxantrone (DHAD,

) (ii) a Epirubicin (Ellence)^TM) (ii) a Idarubicin (A)

) (ii) a Mitomycin C

Geldanamycin; herbimycin; griseofulvin (ravidomycin); and deacetyl griseofulvin (desacetylgriseofulvudomycin).

Exemplary vinblastine alkaloids include, for example, vinorelbine tartrate

Vincristine

And vindesine

) (ii) a Vinblastine (also known as vinblastine sulfate, vinblastine and VLB,

And

) (ii) a And vinorelbine

Exemplary proteasome inhibitors include bortezomib

Carfilzomib (PX-171-007, (S) -4-methyl-N- ((S) -1- (((S) -4-methyl-1- ((R) -2-methyloxiran-2-yl) -1-oxopent-2-yl) amino) -1-oxo-3-phenylpropan-2-yl) -2- ((S) -2- (2-morpholino) -4-phenylbutylamino) -pentanamide); marizomib (NPI-0052); ixazofamid citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N- [ (2-methyl-5-thiazolyl) carbonyl ]-L-seryl-O-methyl-N- [ (1S) -2- [ (2R) -2-methyl-2-oxiranyl]-2-oxo-1- (phenylmethyl) ethyl-L-serine amide (ONX-0912).

In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with berentuzumab (brentuximab). Bernetuzumab is an antibody-drug conjugate of an anti-CD 30 antibody and monomethyl auristatin E. In embodiments, the subject has Hodgkin Lymphoma (HL), e.g., relapsed or refractory HL. In embodiments, the subject comprises CD30+ HL. In embodiments, the subject has received an Autologous Stem Cell Transplant (ASCT). In embodiments, the subject has not received ASCT. In embodiments, the bevacizumab is administered at the following dose: about 1-3mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3mg/kg), e.g., intravenously, e.g., once every 3 weeks.

In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with barbiturate and dacarbazine or in combination with barbiturate and bendamustine. Dacarbazine is an alkylating agent with the chemical name 5- (3, 3-dimethyl-1-triazenyl) imidazole-4-carboxamide. Bendamustine is chemically named 4- [5- [ bis (2-chloroethyl) amino ]-1-methylbenzimidazol-2-yl]An alkylating agent for butyric acid. In embodiments, the subject has Hodgkin's Lymphoma (HL). In embodiments, the subject has not been previously treated with cancer therapy. In embodiments, the subject is at least 60 years of age, e.g., 60, 65, 70, 75, 80, 85 years of age or older. In embodiments, the dacarbazine is administered at the following doses: about 300-450mg/m²(e.g., about 300-325, 325-350, 350-375, 375-400, 400-425, or 425-450mg/m²) For example, intravenous administration. In embodiments, bendamustine is administered at the following doses: about 75-125mg/m²(e.g., 75-100 or 100-125mg/m²For example, about 90mg/m²) For example, intravenous administration. In embodiments, the bevacizumab is administered at the following dose: about 1-3mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3mg/kg), e.g., intravenously, e.g., once every 3 weeks.

In some embodiments, the CAR-expressing cells described herein are administered to a subject in combination with a CD20 inhibitor, e.g., an anti-CD 20 antibody (e.g., an anti-CD 20-specific antibody or bispecific antibody) or fragment thereof. Exemplary anti-CD 20 antibodies include, but are not limited to, rituximab, ofatumumab, ocrelizumab (ocrelizumab), vetuzumab, obinutuzumab, TRU-015 (trubiun Pharmaceuticals), ocatatuzumab, and Pro131921 (Genentech). See, for example, Lim et al, Haematologica.95.1(2010): 135-43.

In some embodiments, the anti-CD 20 antibody comprises rituximab. Rituximab is a chimeric mouse/human monoclonal antibody IgG1 κ that binds to CD20 and causes cell lysis of CD20 expressing cells, e.g., as described in www.accessdata.fda.gov/drug atfda _ docs/label/2010/103705s5311lbl. In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with rituximab. In embodiments, the subject has CLL or SLL.

In some embodiments, rituximab is administered intravenously, e.g., as an intravenous infusion. For example, about 500-2000mg (e.g., about 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, or 1900-2000mg) rituximab per infusion is provided. In some embodiments, rituximab is administered at the following dose: 150mg/m²To 750mg/m²E.g., about 150-175mg/m²、175-200mg/m²、200-225mg/m²、225-250mg/m²、250-300mg/m²、300-325mg/m²、325-350mg/m²、350-375mg/m²、375-400mg/m²、400-425mg/m²、425-450mg/m²、450-475mg/m²、475-500mg/m²、500-525mg/m²、525-550mg/m²、550-575mg/m²、575-600mg/m²、600-625mg/m²、625-650mg/m²、650-675mg/m²Or 675-700mg/m²Wherein m is²Representing the body surface area of the subject. In some embodiments, rituximab is administered at a dosing interval of at least 4 days (e.g., 4, 7, 14, 21, 28, 35 days or longer). For example, rituximab is administered at a dosing interval of at least 0.5 weeks (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8 weeks or longer). In some embodiments, rituximab is administered at a dose and interval as described herein for a period of time, e.g., at least 2 weeks, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more. For example, rituximab is administered at a dose and dosing interval as described herein for a total of at least 4 doses per treatment cycle (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more doses per treatment cycle).

In some embodiments, the anti-CD 20 antibody comprises ofatumumab. Ofatumumab is an anti-CD 20IgG1 kappa human monoclonal antibody with a molecular weight of approximately 149 kDa. For example, alphakaki monoclonal antibodies are generated using transgenic mouse and hybridoma technology and expressed and purified from a recombinant murine cell line (NS 0). See, e.g., www.accessdata.fda.gov/drug atfda _ docs/label/2009/125326 lbl.pdf; and clinical trial identification numbers NCT01363128, NCT01515176, NCT01626352, and NCT 0139591. In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with an ofatumumab. In embodiments, the subject has CLL or SLL.

In some embodiments, the alfuzumab is administered as an intravenous infusion. For example, about 150-3000mg (e.g., about 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1200, 1200-1400, 1400-1600, 1600-1800-2000, 2000-2200, 2200-2400, 2400-2600, 2600-2800, or 2800-3000mg) ofatumumab is provided per infusion. In embodiments, the ofatumumab is administered at an initial dose of about 300mg, followed by 2000mg, e.g., about 11 doses, e.g., for 24 weeks. In some embodiments, ofatumumab is administered at a dosing interval of at least 4 days (e.g., 4, 7, 14, 21, 28, 35 days or longer). For example, ofatumumab is administered at the following dosing intervals: at least 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 26, 28, 20, 22, 24, 26, 28, 30 weeks or more. In some embodiments, ofatumumab is administered at the dosages and dosing intervals described herein for a period of time, e.g., at least 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 40, 50, 60 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, or 1, 2, 3, 4, 5 years or more. For example, ofatumumab is administered at a total of at least 2 doses per treatment cycle (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20 or more doses per treatment cycle) at the doses and dosing intervals described herein.

In some cases, the anti-CD 20 antibody comprises ocrelizumab. Orizumab is a humanized anti-CD 20 monoclonal antibody, e.g., as described in clinical trial identification numbers NCT00077870, NCT01412333, NCT00779220, NCT00673920, NCT01194570, and Kappos et al, Lancet.19.378(2011): 1779-87.

In some cases, the anti-CD 20 antibody comprises veltuzumab. Veltuzumab is a humanized monoclonal antibody directed against CD 20. See, e.g., clinical trial identification numbers NCT00547066, NCT00546793, NCT01101581, and Goldenberg et al, Leuk Lymphoma.51(5) (2010): 747-55.

In some cases, the anti-CD 20 antibody comprises GA 101. GA101 (also known as obinutuzumab or RO5072759) is a humanized and glycoengineered anti-CD 20 monoclonal antibody. See, e.g., Robak. curr. Opin. Investig. drugs.10.6(2009): 588-96: 588-96; the identification number of clinical trials is as follows: NCT01995669, NCT01889797, NCT02229422, and NCT 01414205; and www.accessdata.fda.gov/drug atfda _ docs/label/2013/125486s000lbl.

In some cases, the anti-CD 20 antibody comprises AME-133 v. AME-133v (also known as LY2469298 or ocatatuzumab) is a humanized IgG1 monoclonal antibody directed against CD20 that has increased Fc γ RIIIa receptor affinity and enhanced antibody-dependent cellular cytotoxicity (ADCC) activity compared to rituximab. See, e.g., Robak et al, BioDrugs 25.1(2011): 13-25; and Forero-Torres et al, Clin Cancer Res.18.5(2012): 1395-403.

In some cases, the anti-CD 20 antibody comprises PRO 131921. PRO131921 is a humanized anti-CD 20 monoclonal antibody engineered to have better Fc γ RIIIa binding and enhanced ADCC compared to rituximab. See, e.g., Robak et al, BioDrugs25.1(2011): 13-25; and Casulo et al, Clin Immunol.154.1(2014): 37-46; and clinical trial identification NCT 00452127.

In some cases, the anti-CD 20 antibody comprises TRU-015. TRU-015 is an anti-CD 20 fusion protein derived from the domain of an antibody directed against CD 20. TRU-015 is smaller than monoclonal antibodies, but retains Fc-mediated effector functions. See, e.g., Robak et al, BioDrugs25.1(2011): 13-25. TRU-015 contains a single chain variable fragment (scFv) of anti-CD 20, CH2 and CH3 domains, hingedly linked to human IgG1, but lacks the CH1 and CL domains.

In some embodiments, the anti-CD 20 antibodies described herein are conjugated or conjugated to a therapeutic agent, e.g., a chemotherapeutic agent described herein (e.g., cyclophosphamide, fludarabine, a histone deacetylase inhibitor, a demethylating agent, a peptide vaccine, an antitumor antibiotic, a tyrosine kinase inhibitor, an alkylating agent, an antimicrotubule or an antimitotic agent), an antiallergic agent, an antimigraine (or an antiemetic), an analgesic, or a cytoprotective agent.

In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with a B-cell lymphoma 2(BCL-2) inhibitor (e.g., venetoclax, also known as ABT-199 or GDC-0199) and/or rituximab. In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with venetoclax and rituximab. Venetocalax is a small molecule that inhibits the anti-apoptotic protein BCL-2. The structure of venetoclax (4- (4- { [2- (4-chlorophenyl) -4, 4-dimethylcyclohex-1-en-1-yl ] methyl } piperazin-1-yl) -N- ({ 3-nitro-4- [ (tetrahydro-2H-pyran-4-ylmethyl) amino ] phenyl } sulfonyl) -2- (1H-pyrrolo [2,3-b ] pyridin-5-yloxy) benzamide) is shown below.

In embodiments, the subject has CLL. In embodiments, the subject has relapsed CLL, e.g., the subject has been previously administered cancer therapy. In embodiments, the venetocalax is administered at the following dose: about 15-600mg (e.g., 15-20, 20-50, 50-75, 75-100, 100-200, 200-300, 300-400, 400-500, or 500-600mg), for example, administered once daily. In embodiments, rituximab is administered at the following dose: about 350-550mg/m²(e.g., 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500mg/m ²) E.g., intravenously, e.g., once a month.

In some embodiments, the CAR-expressing cells described herein are administered in combination with an oncolytic virus. In embodiments, the oncolytic virus is capable of selectively replicating in cancer cells and triggering cancer cell death or delaying its growth. In some cases, the oncolytic virus has no or minimal effect on non-cancer cells. Oncolytic viruses include, but are not limited to, oncolytic adenoviruses, oncolytic herpes simplex viruses, oncolytic retroviruses, oncolytic parvoviruses, oncolytic vaccinia viruses, oncolytic sindbis viruses, oncolytic influenza viruses, or oncolytic RNA viruses (e.g., oncolytic reovirus, oncolytic Newcastle Disease Virus (NDV), oncolytic measles virus, or oncolytic Vesicular Stomatitis Virus (VSV)).

In some embodiments, the oncolytic virus is a virus described in US2010/0178684a1, e.g., a recombinant oncolytic virus, which is incorporated herein by reference in its entirety. In some embodiments, the recombinant oncolytic virus comprises a nucleic acid sequence (e.g., a heterologous nucleic acid sequence) encoding an inhibitor of an immune response or inflammatory response, e.g., as described in US2010/0178684a1, which is incorporated herein by reference in its entirety. In embodiments, a recombinant oncolytic virus, e.g., an oncolytic NDV, comprises a pro-apoptotic protein (e.g., an apoptin), a cytokine (e.g., GM-CSF, interferon- γ, interleukin-2 (IL-2), tumor necrosis factor- α), an immunoglobulin (e.g., an antibody against ED-B fibronectin), a tumor associated antigen, a bispecific adapter protein (e.g., a bispecific antibody or antibody fragment directed against NDV HN protein and a T cell co-stimulatory receptor such as CD3 or CD 28; or a fusion protein between human IL-2 and a single chain antibody directed against NDV HN protein). See, e.g., Zamaran et al, Future Microbiol.7.3(2012):347-67, which is incorporated by reference herein in its entirety. In some embodiments, the oncolytic virus is a chimeric oncolytic NDV described in US 8591881B 2, US 2012/0122185 a1, or US 2014/0271677 a1, each of which is incorporated herein by reference in its entirety.

In some embodiments, the oncolytic virus comprises a Conditionally Replicating Adenovirus (CRA) designed to replicate completely in cancer cells. See, for example, Alemany et al, Nature Biotechnol.18(2000): 723-27. In some embodiments, the oncolytic adenovirus comprises the one described in table 1 on page 725 of Alemany et al, which is incorporated herein by reference in its entirety.

Exemplary oncolytic viruses include, but are not limited to, the following:

group B oncolytic adenovirus (ColoAd1) (PsiOxus Therapeutics Ltd.) (see, e.g., clinical trial identification number: NCT 02053220);

ONCOS-102 (formerly CGTG-102), which is an adenovirus containing granulocyte-macrophage colony stimulating factor (GM-CSF) (see, e.g., clinical trial identification number: NCT 01598129);

VCN-01, which is a genetically modified oncolytic human adenovirus encoding human PH20 hyaluronidase (VCN Biosciences, S.L.) (see, e.g., clinical trial identification numbers: NCT02045602 and NCT 02045589);

conditionally replicating adenovirus ICOVIR-5, which is a virus derived from wild-type human adenovirus serotype 5(Had5), has been modified to replicate selectively in cancer cells, with an unregulated retinoblastoma/E2F pathway (Institut cat a d' oncogia) (see, e.g., clinical trial identification number: NCT 01864759);

Celyvir, comprising bone marrow-derived autologous Mesenchymal Stem Cells (MSCs) (Hospital Infantil Universal) infected with oncolytic adenovirus ICOVIR5

Jes U.S., Madrid, Spain/Ramon Alemany) (see, e.g., clinical trial identification numbers: NCT 01844661);

CG0070, a conditionally replicating oncolytic serotype 5 adenovirus (Ad5), in which the human E2F-1 promoter drives expression of essential E1 alphavirus genes, thus limiting viral replication and cytotoxicity to Rb pathway-deficient tumor cells (Cold Genesys, Inc.) (see, e.g., clinical trial identification number: NCT 02143804); or

DNX-2401 (formerly. delta. -24-RGD), which is an adenovirus that has been engineered to selectively replicate in retinoblastoma (Rb) pathway defective cells and to more efficiently infect cells that express certain RGD-binding integrins (Clinica Universal de Navarra, Universal de Navarra/DNATrix, Inc.) (see, e.g., clinical trial identification number: NCT 01956734).

In some embodiments, the oncolytic virus described herein is administered by injection, e.g., subcutaneous, intra-arterial, intravenous, intramuscular, intrathecal, or intraperitoneal injection. In embodiments, the oncolytic virus described herein is administered intratumorally, transdermally, transmucosally, orally, intranasally, or by pulmonary administration.

In one embodiment, the CAR-expressing cells described herein are administered to a subject in combination with a molecule that reduces the population of Treg cells. Methods of reducing the number of Treg cells (e.g., depleting Treg cells) are known in the art and include, for example, CD25 depletion, administration of cyclophosphamide, modulation of GITR function. Without wishing to be bound by theory, it is believed that reducing the number of Treg cells in a subject prior to apheresis or prior to administration of CAR-expressing cells described herein reduces the number of unwanted immune cells (e.g., tregs) in the tumor microenvironment and reduces the risk of relapse in the subject. In one embodiment, the CAR-expressing cells described herein are administered to a subject in combination with a GITR-targeting and/or GITR function-modulating molecule, wherein the GITR-targeting and/or GITR function-modulating molecule is, for example, a GITR agonist and/or a GITR antibody that depletes regulatory T cells (tregs). In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with cyclophosphamide. In one embodiment, the GITR binding molecule and/or a molecule that modulates GITR function (e.g., a GITR agonist and/or a Treg-depleted GITR antibody) is administered prior to administration of the CAR-expressing cells. For example, in one embodiment, a GITR agonist may be administered prior to apheresis of the cells. In embodiments, cyclophosphamide is administered to the subject prior to administration (e.g., infusion or reinfusion) of the CAR-expressing cells or prior to apheresis of the cells. In embodiments, cyclophosphamide and the anti-GITR antibody are administered to the subject prior to administration (e.g., infusion or reinfusion) of the CAR-expressing cells or prior to apheresis of the cells. In one embodiment, the subject has cancer (e.g., a solid cancer or a hematological cancer such as ALL or CLL). In one embodiment, the subject has CLL. In embodiments, the subject has ALL. In embodiments, the subject has a solid cancer, e.g., a solid cancer as described herein. Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as, for example, the GITR fusion proteins described in the following documents; U.S. patent No. 6,111,090, european patent No. 090505B1, U.S. patent No. 8,586,023, PCT publication nos. WO 2010/003118 and 2011/090754, or anti-GITR antibodies such as described in: U.S. patent No. 7,025,962, european patent No. 1947183B1, U.S. patent No. 7,812,135, U.S. patent No. 8,388,967, U.S. patent No. 8,591,886, european patent No. EP 1866339, PCT publication No. WO 2011/028683, PCT publication No. WO 2013/039954, PCT publication No. WO 2005/007190, PCT publication No. WO 2007/133822, PCT publication No. WO 2005/055808, PCT publication No. WO 99/40196, PCT publication No. WO 2001/03720, PCT publication No. WO 99/20758, PCT publication No. WO 2006/083289, PCT publication No. WO 2005/115451, U.S. patent No. 7,618,632, and PCT publication No. WO 2011/051726.

In one embodiment, a CAR-expressing cell described herein is administered to a subject in combination with an mTOR inhibitor (e.g., an mTOR inhibitor described herein, e.g., rapalog such as everolimus). In one embodiment, the mTOR inhibitor is administered prior to the CAR-expressing cells. For example, in one embodiment, the mTOR inhibitor may be administered prior to apheresis of the cells.

In one embodiment, a CAR-expressing cell described herein is administered to a subject in combination with a GITR agonist (e.g., a GITR agonist described herein). In one embodiment, the GITR agonist is administered prior to the CAR-expressing cells. For example, in one embodiment, a GITR agonist may be administered prior to apheresis of the cells.

In one embodiment, a CAR-expressing cell described herein is administered to a subject in combination with a protein tyrosine phosphatase inhibitor (e.g., a protein tyrosine phosphatase inhibitor described herein). In one embodiment, the protein tyrosine phosphatase inhibitor is an SHP-1 inhibitor, e.g., an SHP-1 inhibitor described herein, such as, for example, antimony gluconate. In one embodiment, the protein tyrosine phosphatase inhibitor is a SHP-2 inhibitor.

In one embodiment, the CAR-expressing cells described herein can be used in combination with a kinase inhibitor. In one embodiment, the kinase inhibitor is a CDK4 inhibitor (e.g., a CDK4 inhibitor described herein, e.g., a CD4/6 inhibitor, such as, e.g., 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2,3-d ] pyrimidin-7-one hydrochloride (also known as pabulib or PD0332991) in one embodiment, the kinase inhibitor is a BTK inhibitor, e.g., a BTK inhibitor described herein, such as, e.g., ibrutinib in one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitor described herein, such as, e.g., rapamycin, a rapamycin analogue, OSI-027. the mTOR inhibitor can, e.g., be an mTORC1 inhibitor and/or mTORC2 inhibitor, for example, mTORC1 inhibitors and/or mTORC2 inhibitors described herein. In one embodiment, the kinase inhibitor is a MNK inhibitor, e.g., a MNK inhibitor described herein, such as, e.g., 4-amino-5- (4-fluoroanilino) -pyrazolo [3,4-d ] pyrimidine. The MNK inhibitor may, for example, be an MNK1a, MNK1b, MNK2a and/or MNK2b inhibitor. In one embodiment, the kinase inhibitor is a duplex PI3K/mTOR inhibitor as described herein, such as, for example, PF-04695102. In one embodiment, the kinase inhibitor is a DGK inhibitor, e.g., a DGK inhibitor described herein, such as, e.g., DGKinh1(D5919) or DGKinh2 (D5794).

In one embodiment, the kinase inhibitor is a CDK4 inhibitor selected from: aloisine A; froralpididol or HMR-1275, 2- (2-chlorophenyl)-5, 7-dihydroxy-8- [ (3S,4R) -3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone; crizotinib (PF-02341066); 2- (2-chlorophenyl) -5, 7-dihydroxy-8- [ (2R,3S) -2- (hydroxymethyl) -1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one, hydrochloride (P276-00); 1-methyl-5- [ [2- [5- (trifluoromethyl) -1H-imidazol-2-yl]-4-pyridinyl]Oxygen gas]-N- [4- (trifluoromethyl) phenyl]-1H-benzimidazol-2-amine (RAF 265); indesulam (E7070); roscovitine (CYC 202); palbociclib (PD 0332991); dinaciclib (SCH 727965); n- [5- [ [ (5-tert-butyl)

Oxazol-2-yl) methyl]Thio group]Thiazol-2-yl]Piperidine-4-carboxamide (BMS 387032); 4- [ [ 9-chloro-7- (2, 6-difluorophenyl) -5H-pyrimido [5,4-d ]][2]Benzazepin-2-yl]Amino group]-benzoic acid (MLN 8054); 5- [3- (4, 6-difluoro-1H-benzimidazol-2-yl) -1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine (AG-024322); 4- (2, 6-dichlorobenzamido) -1H-pyrazole-3-carboxylic acid N- (piperidin-4-yl) amide (AT 7519); 4- [ 2-methyl-1- (1-methylethyl) -1H-imidazol-5-yl]-N- [4- (methylsulfonyl) phenyl ]-2-pyrimidineamine (AZD5438) and XL281(BMS 908662).

In one embodiment, the kinase inhibitor is a CDK4 inhibitor, e.g., palbociclib (PD0332991), and palbociclib is administered at a dose of about 50mg, 60mg, 70mg, 75mg, 80mg, 90mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg (e.g., 75mg, 100mg, or 125mg) daily for a period of time, e.g., daily for 14-21 days of a 28-day cycle, or daily for 7-12 days of a 21-day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of palbociclib are administered.

In embodiments, the CAR-expressing cells described herein are administered to the subject in combination with a cyclin-dependent kinase (CDK)4 or 6 inhibitor (e.g., a CDK4 inhibitor or a CDK6 inhibitor described herein). In embodiments, the CAR-expressing cells described herein are administered to the subject in combination with a CDK4/6 inhibitor (e.g., an inhibitor that targets both CDK4 and CDK 6) (e.g., a CDK4/6 inhibitor described herein). In one embodiment, the subject has MCL. MCL is an aggressive cancer that responds poorly (i.e., is essentially incurable) to currently available therapies. In many cases of MCL, cyclin D1 (a regulatory protein of CDK 4/6) is expressed in MCL cells (e.g., due to chromosomal translocations involving immunoglobulin genes and cyclin D1 genes). Thus, without being bound by theory, MCL cells are believed to be highly sensitive and specific for CDK4/6 inhibition (i.e., have minimal impact on normal immune cells). CDK4/6 inhibitor alone had some efficacy in treating MCL, but only partial remission was achieved with high recurrence rate. An exemplary CDK4/6 inhibitor is LEE011 (also known as ribociclib), the structure of which is shown below.

Without being bound by theory, it is believed that administration of a CAR-expressing cell described herein in combination with a CDK4/6 inhibitor (e.g., LEE011 or other CDK4/6 inhibitors described herein) can achieve higher reactivity, e.g., higher remission rate and/or lower relapse rate, as compared to administration of CDK4/6 inhibitor alone.

In one embodiment, the kinase inhibitor is a BTK inhibitor selected from the group consisting of: ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In a preferred embodiment, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2 inducible kinase (ITK) and is selected from the group consisting of GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13.

In one embodiment, the kinase inhibitor is a BTK inhibitor, e.g., ibrutinib (PCI-32765). In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with a BTK inhibitor (e.g., ibrutinib). In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with ibrutinib (also known as PCI-32765). The structure of ibrutinib (1- [ (3R) -3- [ 4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl ] piperidin-1-yl ] prop-2-en-1-one) is shown below.

In embodiments, the subject has CLL, Mantle Cell Lymphoma (MCL), or Small Lymphocytic Lymphoma (SLL). For example, the subject has a deletion (del (17p) in the short arm of chromosome 17 (e.g., in leukemia cells), in other examples, the subject does not have a del (17p), in embodiments, the subject has relapsed CLL or SLL, e.g., has previously been administered one cancer therapy (e.g., has previously been administered one, two, three, or four prior cancer therapies), in embodiments, the subject has refractory CLL or SLL in other embodiments, the subject has follicular lymphoma, e.g., relapsed or refractory follicular lymphoma, in embodiments, ibrutinib is administered at a dose of about 300-600 mg/day (e.g., about 300-350, 350-400, 400-450, 450-500-550, or 550-600 mg/day, e.g., about 420 mg/day or about 560 mg/day), for example, oral administration. In embodiments, ibrutinib is administered at a dose of about 250mg, 300mg, 350mg, 400mg, 420mg, 440mg, 460mg, 480mg, 500mg, 520mg, 540mg, 560mg, 580mg, 600mg (e.g., 250mg, 420mg, or 560mg) daily for a period of time, e.g., daily for a 21 day period, or daily for a 28 day period. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of ibrutinib are administered. In some embodiments, ibrutinib is administered in combination with rituximab. See, for example, Burger et al, (2013) Ibrutinib In Combination With Rituximab (iR) Is Well-treated and indexes a High Rate Of Dual detectable dependencies In Patients With High-Risk Chronic reactivity Leukaemia (CLL): New, Updated Results Of a Phase II Trial In 40 Patients, abstract 675, provided at 55 th ASH annular Meeting and Exposure, New Orleanes, LA 7-10 Dec. Without being bound by theory, it is believed that the addition of ibrutinib enhances the T cell proliferative response and may switch T cells from a helper T-2(Th2) phenotype to a helper T-1(Th1) phenotype. Th1 and Th2 are helper T cell phenotypes in which Th1 and Th2 direct different immune response pathways. The Th1 phenotype is associated with a pro-inflammatory response, e.g., for killing cells, such as intracellular pathogens/viruses or cancer cells, or perpetuating an autoimmune response. The Th2 phenotype is associated with eosinophil accumulation and an anti-inflammatory response.

In some embodiments of the methods, uses and compositions herein, the BTK inhibitor is a BTK inhibitor described in international application WO/2015/079417, which is incorporated herein by reference in its entirety. For example, in some embodiments, the BTK inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof;

wherein the content of the first and second substances,

r1 is hydrogen, C1-C6 alkyl optionally substituted with hydroxy;

r2 is hydrogen or halogen;

r3 is hydrogen or halogen;

r4 is hydrogen;

r5 is hydrogen or halogen;

or R4 and R5 are linked to each other and represent a bond, -CH2-, -CH2-CH2-, -CH ═ CH-CH 2-; -CH2-CH ═ CH-; or-CH 2-CH2-CH 2-;

r6 and R7 independently of one another represent H, C1-C6 alkyl optionally substituted by hydroxy, C3-C6 cycloalkyl optionally substituted by halogen or hydroxy, or halogen;

r8, R9, R', R10 and R11 independently of one another represent H or C1-C6 alkyl optionally substituted by C1-C6 alkoxy; or any two of R8, R9, R, R', R10, and R11 together with the carbon atoms to which they are bound may form a 3-6 membered saturated carbocyclic ring;

r12 is hydrogen or C1-C6 alkyl optionally substituted with halogen or C1-C6 alkoxy;

or R12 and any one of: r8, R9, R, R', R10 or R11 together with the atoms to which they are bound may form a 4, 5, 6 or 7 membered nitrogen heterocycle, which ring may be optionally substituted by halogen, cyano, hydroxy, C1-C6 alkyl or C1-C6 alkoxy;

n is 0 or 1; and is

R13 is C2-C6 alkenyl optionally substituted with C1-C6 alkyl, C1-C6 alkoxy, or N, N-di-C1-C6 alkylamino; C2-C6 alkynyl optionally substituted with C1-C6 alkyl or C1-C6 alkoxy; or C2-C6 alkylene oxides (C2-C6 alkylenyl oxides) optionally substituted with C1-C6 alkyl.

In some embodiments, the BTK inhibitor of formula I is selected from: n- (3- (5- ((1-acryloyloxyazetidin-3-yl) oxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; ((E) -N- (3- (6-amino-5- ((1- (but-2-enoyl) azetidin-3-yl) oxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide, ((3- (6-amino-5- ((1-propynylazetidin-3-yl) oxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide), N- (3- (6-amino-5- ((1- (but-2-propynylazetidin-3-yl) oxy) pyrimidin-4-yl) -5-fluoro-2-fluorobenzamide -2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; n- (3- (5- ((1-acryloyloxypiperidin-4-yl) oxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; n- (3- (6-amino-5- (2- (N-methacrylamido) ethoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; ((E) -N- (3- (6-amino-5- (2- (N-methylbut-2-enylamino) ethoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide, ((E) -N- (3- (6-amino-5- (2- (N-methylpropynylamino) ethoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; ((E) -N- (3- (6-amino-5- (2- (4-methoxy-N-methylbut-2-enylamino) ethoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; n- (3- (6-amino-5- (2- (N-methylbut-2-ynylamino) ethoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; n- (2- ((4-amino-6- (3- (4-cyclopropyl-2-fluorobenzamide) -5-fluoro-2-methylphenyl) pyrimidin-5-yl) oxy) ethyl) -N-methyloxirane-2-carboxamide; n- (2- ((4-amino-6- (3- (6-cyclopropyl-8-fluoro-1-oxoisoquinolin-2 (1H) -yl) phenyl) pyrimidin-5-yl) oxy) ethyl) -N-methacrylamide; n- (3- (5- (2-acrylamidoethoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; n- (3- (6-amino-5- (2- (N-ethylacrylamide) ethoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; n- (3- (6-amino-5- (2- (N- (2-fluoroethyl) acrylamide) ethoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; n- (3- (5- ((1-acrylamidocyclopropyl) methoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; ((S) -N- (3- (5- (2-acrylamidopropoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; ((S) -N- (3- (6-amino-5- (2- (but-2-ynylamino) propoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; ((S) -N- (3- (6-amino-5- (2- (N-methacrylamido) propoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) - 4-cyclopropyl-2-fluorobenzamide; ((S) -N- (3- (6-amino-5- (2- (N-methylbut-2-ynylamino) propoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide, ((S) -N- (3- (5- ((1-acryloyloxypyrrolidin-2-yl) methoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; (S) -N- (3- (5- ((1-acryloyloxypyrrolidin-2-yl) methoxy) -6-aminopyrimidin-4-yl) -5-fluoro- 2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; ((S) -N- (3- (6-amino-5- ((1- (but-2-ynoylpyrrolidin-2-yl) methoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide ((S) -2- (3- (5- ((1-acryloyloxypyrrolidin-2-yl) methoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2- (hydroxymethyl) phenyl) -6-cyclopropyl-3, 4-dihydroisoquinolin-1 (2H) -one, N- (2- ((4-amino-6- (3- (6-cyclopropyl-1-oxy) phenyl) 3, 4-dihydro-2 (1H) -5-fluoro-2- (hydroxymethyl) phenyl) pyrimidin-5-yl) oxy) ethyl-N-methylacrylamide; n- (3- (5- (((2S,4R) -1-acryloyloxy-4-methoxypyrrolidin-2-yl) methoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; n- (3- (6-amino-5- (((2S,4R) -1- (but-2-ynoyl-4-methoxypyrrolidin-2-yl) methoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide, 2- (3- (5- (((2S,4R) -1-acryloyloxy-4-methoxypyrrolidin-2-yl) methoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2- (hydroxymethyl) phenyl) -6-cyclopropyl-3, 4-dihydroisoquinolin-1 (2H) -one, N- (3- (5- (((2S,4R) -1- (2S-methoxy-pyrrolidin-2-yl) methoxy) pyrimidin-4-yl) -5-fluoro-2- (hydroxymethyl) phenyl) -6-cyclopropyl-3, 4-dihydroisoquinolin-1 (2H) -one 4S) -1-acryloyloxy-4-methoxypyrrolidin-2-yl) methoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; n- (3- (6-amino-5- (((2S,4S) -1- (but-2-ynoyl-4-methoxypyrrolidin-2-yl) methoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide, N- (3- (5- (((2S,4R) -1-acryloyloxy-4-fluoropyrrolidin-2-yl) methoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide, N- (3- (6-amino-5- (((2S,4R) -1- (but-2-ynoyl-4-fluoropyrrolidin-2-yl) methoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; ((S) -N- (3- (5- ((1-acryloyloxyazetidin-2-yl) methoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; ((S) -N- (3- (6-amino-5- ((1-propioylazetidin-2-yl) methoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; ((S) -2- (3- (5- ((1-acryloyloxyazetidin-2-yl) methoxy) -6-aminopyrimidin-4-yl) - 5-fluoro-2- (hydroxymethyl) phenyl) -6-cyclopropyl-3, 4-dihydroisoquinolin-1 (2H) -one; ((R) -N- (3- (5- ((1-acryloyloxyazetidin-2-yl) methoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide ((R) -N- (3- (5- ((1-acryloyloxypiperidin-3-yl) methoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N- (3- (5- (((2R,3S) -1-acryloyloxy-3-methoxypyrrolidin-2-yl) methoxy) -6-aminopyrimidine Pyridin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; n- (3- (5- (((2S,4R) -1-acryloyloxy-4-cyanopyrrolidin-2-yl) methoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; or N- (3- (5- (((2S,4S) -1-acryloyloxy-4-cyanopyrrolidin-2-yl) methoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide.

Unless otherwise provided, the chemical terms used above in describing the BTK inhibitors of formula I are used according to their meaning as described in international application WO/2015/079417, which is incorporated herein in its entirety by reference.

In one embodiment, the kinase inhibitor is an mTOR inhibitor selected from the group consisting of: tacrolimus(ii) a Dipholimus ((1R,2R,4S) -4- [ (2R) -2[ (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R) -1, 18-dihydroxy-19, 30-dimethoxy-15, 17,21,23,29, 35-hexamethyl-2, 3,10,14, 20-pentaoxo-11, 36-dioxa-4-azatricyclo [30.3.1.0^4,9]Thirty-seven carbon-16, 24,26, 28-tetraen-12-yl]Propyl radical]2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK 8669; everolimus (RAD 001); rapamycin (AY 22989); samimod (simapimod); (5- {2, 4-bis [ (3S) -3-methylmorpholin-4-yl)]Pyrido [2,3-d]Pyrimidin-7-yl } -2-methoxyphenyl) methanol (AZD 8055); 2-amino-8- [ trans-4- (2-hydroxyethoxy) cyclohexyl]-6- (6-methoxy-3-pyridyl) -4-methyl-pyrido [2,3-d]Pyrimidin-7 (8H) -one (PF 04691502); and N²- [1, 4-dioxo-4- [ [4- (4-oxo-8-phenyl-4H-1-benzopyran-2-yl) morpholinium-4-yl]]Methoxy radical ]Butyl radical]-L-arginylglycyl-L-alpha-aspartylL-serine- (SEQ ID NO:378), inner salt (SF 1126); and XL 765.

In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., rapamycin, and rapamycin is administered at a dose of about 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg (e.g., 6mg) daily for a period of time, e.g., daily for a 21 day cycle, or daily for a 28 day cycle. In one embodiment, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of rapamycin are administered. In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., everolimus, and everolimus is administered at a dose of about 2mg, 2.5mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg (e.g., 10mg) daily for a period of time, e.g., daily for a 28 day period. In one embodiment, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of everolimus are administered.

In one embodiment, the kinase inhibitor is a MNK inhibitor selected from the group consisting of: CGP 052088; 4-amino-3- (p-fluorophenylamino) -pyrazolo [3,4-d ] pyrimidine (CGP 57380); cercosporamide; ETC-1780445-2; and 4-amino-5- (4-fluoroanilino) -pyrazolo [3,4-d ] pyrimidine.

In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with a phosphatidylinositol 3-kinase (PI3K) inhibitor (e.g., a PI3K inhibitor described herein, e.g., gillidide (idelalisib) or duvelisib) and/or rituximab. In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with girlidide and rituximab. In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with duvelisib and rituximab. Gilidard (also known as GS-1101 or CAL-101; Gilead) is a small molecule that blocks the PI3K delta isoform. The structure of girlidide (5-fluoro-3-phenyl-2- [ (1S) -1- (7H-purin-6-ylamino) propyl ] -4(3H) -quinazolinone) is shown below.

Duvelisib (also known as IPI-145; Infinity Pharmaceuticals and Abbvie) is a small molecule that blocks PI3K- δ, γ. The structure of duvelisib (8-chloro-2-phenyl-3- [ (1S) -1- (9H-purin-6-ylamino) ethyl ] -1(2H) -isoquinolinone) is shown below.

In embodiments, the subject has CLL. In embodiments, the subject has relapsed CLL, e.g., has been previously administered cancer therapy to the subject (e.g., has been previously administered an anti-CD 20 antibody or has been previously administered ibrutinib). For example, the subject has a deletion in the short arm of chromosome 17 (del (17p) (e.g., in leukemia cells) — in other examples, the subject does not del (17p) — in embodiments, the subject is comprised of an immunoglobulin heavy chain variable region (IgV) _H) Leukemia cells comprising a mutation in the gene. In other embodiments, the subject does not comprise an immunoglobulin heavy chain variable region (IgV)_H) Leukemia cells comprising a mutation in the gene. In embodiments, the subject has a deletion in the long arm of chromosome 11 (del (11 q)). In other embodiments, the subject does not have del (1)1 q). In embodiments, girard is administered at the following dose: about 100-. In embodiments, duvelisib is administered at the following dose: about 15-100mg (e.g., about 15-25, 25-50, 50-75, or 75-100mg), for example, 2 administrations per day. In embodiments, rituximab is administered at the following dose: about 350-550mg/m²(e.g., 350-²) For example, intravenous administration.

In one embodiment, the kinase inhibitor is a dual phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitor selected from the group consisting of: 2-amino-8- [ trans-4- (2-hydroxyethoxy) cyclohexyl]-6- (6-methoxy-3-pyridyl) -4-methyl-pyrido [2,3-d ]Pyrimidin-7 (8H) -one (PF-04691502); n- [4- [ [4- (dimethylamino) -1-piperidinyl group]Carbonyl radical]Phenyl radical]-N' - [4- (4, 6-di-4-morpholinyl-1, 3, 5-triazin-2-yl) phenyl]Urea (PF-05212384, PKI-587); 2-methyl-2- {4- [ 3-methyl-2-oxo-8- (quinolin-3-yl) -2, 3-dihydro-1H-imidazo [4,5-c]Quinolin-1-yl]Phenyl } propionitrile (BEZ-235); apitolisib (GDC-0980, RG 7422); 2, 4-difluoro-N- {2- (methoxy) -5- [4- (4-pyridazinyl) -6-quinolinyl]-3-pyridinyl } benzenesulfonamide (GSK 2126458); 8- (6-methoxy-3-yl) -3-methyl-1- (4- (piperazin-1-yl) -3- (trifluoromethyl) phenyl) -1H-imidazo [4,5-c]Quinolin-2 (3H) -one maleic acid (NVP-BGT 226); 3- [4- (4-Morpholinylpyrido [3',2':4,5 ]]Furo [3,2-d ] s]Pyrimidin-2-yl]Phenol (PI-103); 5- (9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl) pyrimidin-2-amine (VS-5584, SB 2343); and N- [2- [ (3, 5-dimethoxyphenyl) amino group]Quinoline derivatives

Lin-3-yl radical]-4- [ (4-methyl-3-methoxyphenyl) carbonyl]Aminobenzenesulfonamide (XL 765).

In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with an Anaplastic Lymphoma Kinase (ALK) inhibitor. Exemplary ALK kinase inhibitors include, but are not limited to, crizotinib (Pfizer), ceritinib (Novartis), oletinib (aletinib) (Chugai), brigatinib (also known as AP 26113; Ariad), entretinib (ignyta), PF-06463922(Pfizer), TSR-011(Tesaro) (see, e.g., clinical trial identification number NCT02048488), CEP-37440(Teva), and X-396 (Xcovery). In some embodiments, the subject has a solid cancer, e.g., a solid cancer described herein, e.g., lung cancer.

The chemical name of crizotinib is 3- [ (1R) -1- (2, 6-dichloro-3-fluorophenyl) ethoxy]-5- (1-piperidin-4-ylpyrazol-4-yl) pyridin-2-amine. The chemical name of ceritinib is 5-chloro-N²- [ 2-isopropoxy-5-methyl-4- (4-piperidinyl) phenyl group]-N⁴- [2- (isopropylsulfonyl) phenyl]-2, 4-pyrimidinediamine. The chemical name of the Alletinib is 9-ethyl-6.6-dimethyl-8- (4-morpholinopiperidin-1-yl) -11-oxo-6, 11-dihydro-5H-benzo [ b]Carbazole-3-carbonitrile. The chemical name of brigitanib is 5-chloro-N²- {4- [4- (dimethylamino) -1-piperidinyl group]-2-methoxyphenyl } -N⁴- [2- (Dimethylphosphoryloxyphenyl) phenyl]-2, 4-pyrimidine. The chemical name of entretinib is N- (5- (3, 5-difluorobenzyl) -1H-indazol-3-yl) -4- (4-methylpiperazin-1-yl) -2- ((tetrahydro-2H-pyran-4-yl) amino) benzamide. PF-06463922 is chemically (10R) -7-amino-12-fluoro-2, 10, 16-trimethyl-15-oxo-10, 15,16, 17-tetrahydro-2H-8, 4- (methano) pyrazolo [4,3-H][2,5,11]-benzo (b)

Diazatetetradecyl (slow) ring-3-carbonitrile. The chemical structure of CEP-37440 is (S) -2- ((5-chloro-2- ((6- (4- (2-hydroxyethyl) piperazin-1-yl) -1-methoxy-6, 7,8, 9-tetrahydro-5H-benzo [ 7)]Cyclohepten-2-yl) amino) pyrimidin-4-yl) amino) -N-methylbenzamide. The chemical name of X-396 is (R) -6-amino-5- (1- (2, 6-dichloro-3-fluorophenyl) ethoxy) -N- (4- (4-methylpiperazine-1-carbonyl) phenyl) pyridazine-3-carboxamide.

Drugs that inhibit the calcineurin of the calcium-dependent phosphatase (cyclosporin and FK506) or the p70S6 kinase important for growth factor-induced signaling (rapamycin) can also be used (Liu et al, Cell 66:807-815, 1991; Henderson et al, Immun.73:316-321, 1991; Bierer et al, curr. Opin. Immun.5:763-773, 1993). In yet another aspect, the cell compositions of the invention may be administered to a subject (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation, T cell ablation therapy with a chemotherapeutic agent, such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, and/or an antibody, such as OKT3 or CAMPATH. In one aspect, the cell composition of the invention is administered following a B-cell ablation therapy such as a drug that reacts with CD20 (e.g., Rituxan). For example, in one embodiment, the subject may receive standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following transplantation, the subject receives an infusion of expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered before or after surgery.

In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with an indoleamine 2, 3-dioxygenase (IDO) inhibitor. IDO is an enzyme that catalyzes the degradation of the amino acid L-tryptophan to kynurenine. IDO is overexpressed by many cancers, for example, prostate, colorectal, pancreatic, cervical, gastric, ovarian, head, and lung cancers. pdcs, macrophages and Dendritic Cells (DCs) can express IDO. Without being bound by theory, it is believed that the (e.g., IDO catalyzed) reduction of L-tryptophan produces an immunosuppressive environment by inducing T cell disability and apoptosis. Thus, without being bound by theory, it is believed that IDO inhibitors can enhance the efficacy of CAR-expressing cells described herein, e.g., by reducing inhibition or reducing death of CAR-expressing immune cells. In embodiments, the subject has a solid tumor, e.g., a solid tumor described herein, e.g., prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, gastric cancer, ovarian cancer, head cancer, or lung cancer. Exemplary IDO inhibitors include, but are not limited to, 1-methyl-tryptophan, indoximod (new link genetics) (see, e.g., clinical trial identification number NCT 01191216; NCT01792050), and INCB024360(Incyte Corp.) (see, e.g., clinical trial identification number NCT 01604889; NCT01685255)

In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with a regulator of myeloid-derived suppressor cells (MDSCs). MDSCs accumulate around many solid tumors and at their tumor sites. These cells inhibit T cell responses, thus hampering the efficacy of cell therapies expressing CARs. Without being bound by theory, it is believed that administration of the MDSC modulator enhances the potency of the CAR-expressing cells described herein. In one embodiment, the subject has a solid tumor, e.g., a solid tumor as described herein, e.g., a glioblastoma. Exemplary MDSC modulators include, but are not limited to, MCS110 and BLZ 945. MCS110 is a monoclonal antibody (mAb) against macrophage colony stimulating factor (M-CSF). See, for example, clinical trial identification number NCT 00757757. BLZ945 is a small molecule inhibitor of colony stimulating factor 1 receptor (CSF 1R). See, e.g., Pyonteck et al, nat. Med.19(2013): 1264-72. The structure of BLZ945 is shown below.

In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with CD19 CART cells (e.g., CTL019, e.g., as described in WO2012/079000, which is incorporated herein by reference). In embodiments, the subject has Acute Myeloid Leukemia (AML), e.g., CD 19-positive AML or CD 19-negative AML. In embodiments, the subject has a CD19+ lymphoma, e.g., CD19+ non-hodgkin lymphoma (NHL), CD19+ FL, or CD19+ DLBCL. In embodiments, the subject has relapsed or refractory CD19+ lymphoma. In embodiments, the lymphocyte-depleting chemotherapy is administered to the subject prior to, concurrently with, or after the administration of the CD19 CART cells. In one example, a lymphocyte-depleting chemotherapy is administered to the subject prior to administration of the CD19 CART cells. For example, lymphocyte depleting chemotherapy is terminated 1-4 days (e.g., 1, 2, 3, or 4 days) prior to CD19 CAR cell infusion. In embodiments, for example, as described herein, multiple doses of CD19 CART cells are administered. For example, a single dose contains about 5x10 ⁸A CD19 CART cell. In embodiments, the lymphocyte-depleting chemotherapy is prior to, concurrent with, or administered to a CAR-expressing cell (e.g., a non-CD 19 CAR-expressing cell) described hereinFollowed by administration to the subject. In embodiments, the CD19 CART is administered to the subject prior to, concurrently with, or subsequent to the administration of a non-CD 19 CAR expressing cell (e.g., a non-CD 19 CAR expressing cell described herein).

In some embodiments, the CAR-expressing cells described herein are administered to a subject in combination with CD19 CAR-expressing cells (e.g., CTL019, e.g., CTL019 as described in WO 2012/079000), which are incorporated herein by reference, to treat a disease associated with expression of CD33, e.g., a cancer described herein. Without being bound by theory, it is believed that administration of cells expressing CD19 CAR in combination with cells expressing CAR improves the efficacy of the CAR-expressing cells described herein by targeting early lineage cancer cells (e.g., cancer stem cells), modulating immune responses, depleting regulatory B cells, and/or improving the tumor microenvironment. For example, cells expressing a CD19 CAR target cancer cells expressing early lineage markers (e.g., cancer stem cells) and cells expressing CD19, while cells expressing a CAR described herein target cancer cells expressing late lineage markers, e.g., CD 33. This priming regimen can improve the efficacy of the CAR-expressing cells described herein. In embodiments, the CD19 CAR-expressing cell is administered prior to, concurrently with, or after administration of the CAR-expressing cell described herein.

In embodiments, the CAR-expressing cells described herein further express a CD 19-targeted CAR, e.g., a CD19 CAR. In one embodiment, a CAR-expressing cell described herein and a CD19 CAR are administered to a subject to treat a cancer described herein, e.g., AML. In one embodiment, the layout of one or both CAR molecules comprises a primary intracellular signaling domain and a costimulatory signaling domain. In another embodiment, the layout of one or both CAR molecules comprises a primary intracellular signaling domain and two or more (e.g., 2, 3, 4, or 5 or more) costimulatory signaling domains. In such embodiments, the CAR molecule and the CD19 CAR described herein can have the same or different primary intracellular signaling domains, the same or different costimulatory signaling domains, or the same or different numbers of costimulatory signaling domains. Alternatively, the CAR and CD19 CAR described herein are configured as a split CAR, wherein one CAR molecule comprises an antigen binding domain and a costimulatory domain (e.g., 4-1BB) and the other CAR molecule comprises an antigen binding domain and a primary intracellular signaling domain (e.g., CD3 ζ).

In some embodiments, the CAR-expressing cells described herein are administered to a subject in combination with an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide (e.g., human Therapeutics, LLC). hetIL-15 is a heterodimeric, non-covalent complex of IL-15 and IL-15 Ra. hetIL-15 is described, for example, in US 8,124,084, US 2012/0177598, US 2009/0082299, US 2012/0141413, and US 2011/0081311, which are incorporated herein by reference. In embodiments, het-IL-15 is administered subcutaneously. In embodiments, the subject has a cancer, e.g., a solid cancer, e.g., melanoma or colon cancer. In embodiments, the subject has metastatic cancer.

In embodiments, a subject having a disease described herein (e.g., a hematological disease, e.g., AML or MDS) is administered a CAR-expressing cell described herein in combination with a drug (e.g., a cytotoxic or chemotherapeutic drug, a biotherapeutic drug (e.g., an antibody, e.g., a monoclonal antibody or cytotherapeutic drug), or an inhibitor (e.g., a kinase inhibitor)). In embodiments, a subject is administered a CAR-expressing cell described herein in combination with a cytotoxic drug (e.g., CPX-351 (cell Pharmaceuticals), cytarabine, zorubicin, vosaroxin (sunsides Pharmaceuticals), sapacitabine (cyclacell Pharmaceuticals), idarubicin, or mitoxantrone). CPX-351 is a liposomal formulation comprising cytarabine and zorubicin in a 5:1 molar ratio. In embodiments, a subject is administered a CAR-expressing cell described herein in combination with a hypomethylation drug (e.g., a DNA methyltransferase inhibitor, e.g., azacitidine or decitabine). In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with a biotherapeutic (e.g., an antibody or cell therapy, e.g., 225 Ac-lintuzumab (lintuzumab) (Actimab-A; Actinium Pharmaceuticals), IPH2102(Innate Pharma/Bristol Myers Squibb), SGN-CD33A (Seattle Genetics), or gemtuzumab ozogamicin (Mylotarg; Pfizer)). SGN-CD33A is an antibody-drug conjugate (ADC) comprising a pyrrolobenzodiazepine dimer linked to an anti-CD 33 antibody. Actimab-A is an actinium-labeled anti-CD 33 antibody (lintuzumab). IPH2102 is a monoclonal antibody targeted to kill immunoglobulin-like receptors (KIRs). In embodiments, a subject is administered a CAR-expressing cell described herein in combination with a FLT3 inhibitor (e.g., sorafenib (Bayer), midostaurin (midostaurin) (Novartis), quinzatinib (quinzartinib) (Daiichi Sankyo), crenolanib (acid Pharmaceuticals), PLX3397(Daiichi Sankyo), AKN-028(Akinion Pharmaceuticals), or ASP2215 (Astellas)). In embodiments, a subject is administered a CAR-expressing cell described herein in combination with an Isocitrate Dehydrogenase (IDH) inhibitor (e.g., AG-221(Celgene/Agios) or AG-120 (Agios/Celgene)). In embodiments, the subject is administered a cell expressing a CAR described herein in combination with an inhibitor of a cell cycle regulator (e.g., polo-like kinase 1(Plk1), e.g., volasertib (Boehringer Ingelheim), or an inhibitor of cyclin-dependent kinase 9(Cdk9), e.g., Alleno Pharmaceuticals/Sanofi Aventis, in embodiments, the subject is administered a cell expressing a CAR described herein in combination with an inhibitor of a B-cell receptor signaling network inhibitor, e.g., B-cell lymphoma 2(Bcl-2), e.g., Phaveteocalax (Abbvie/Roche), or an inhibitor of Bruton tyrosine kinase (Btk), e.g., Eilutinib (Pharmaceuticals/Johnson & Johnson Jansen Pharmaceutical), in embodiments, the CAR is administered to the subject in combination with an inhibitor of a cell cycle, e.g., a lipo aminopeptidase (HDAC) inhibitor, e.g., a histone deacetylase (HDAC 1), pracinostat (mei pharma); multiple kinase inhibitors (e.g., rigosertib (Onconova Therapeutics/Baxter/SymBio)); or a peptide CXCR4 inverse agonist (e.g., BL-8040(BioLineRx)) in combination with a CAR-expressing cell described herein. In embodiments, CD 33-targeted CAR-expressing cells are administered to a subject in combination with CAR-expressing cells that target antigens other than CD33 (e.g., CLL, BCMA, CD123, CD19, FLT-3, or folate receptor beta).

In another embodiment, the subject receives an infusion of the CART33 cell composition of the invention prior to transplantation of the cells (e.g., an allogeneic stem cell graft). In a preferred embodiment, the CART33 cells transiently express CAR33, e.g., by electroporation of mRNA anti-CD 33 CAR, thereby terminating expression of CAR33 prior to infusion of the donor stem cells to avoid implantation failure.

Some patients may develop allergic reactions to the compounds of the present invention and/or other anticancer drugs during or after administration; therefore, anti-allergic drugs are often administered to minimize the risk of allergic reactions. Suitable anti-allergic agents include corticosteroids, such as dexamethasone (e.g.,

) The compound (a) is a compound of beclomethasone (e.g.,

) Hydrocortisone (also known as cortisone, sodium hydrocortisone succinate, sodium hydrocortisone phosphate and under the trade name hydrocortisone sodium succinate)

The amount of hydrocortisone phosphate to be added,

Hydrocort

and

sold), prednisolone (prednisolone) (under the trade name prednisolone)

And

sold), prednisone (under the trade name prednisone)

Liquid

And

sold), methylprednisolone (also known as 6-methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate under the trade name

And

sold); antihistamines, such as diphenhydramine (e.g.,

) Clotrimine and cyproheptadine; and bronchodilators, such as beta-adrenergic receptor agonists, albuterol (albuterol) (e.g.,

) And terbutaline

Some patients may experience nausea during and after administration of the compounds of the present invention and/or other anti-cancer agents; thus, the use of anti-emetics prevents nausea (regurgitation) and vomiting. Suitable anti-emetics include aprepitant

Ondansetron

HCl granisetron

Lorazepam

Dexamethasone

Ethanedisulfonic acid

Casopitant (C)

And

) And combinations thereof.

During the treatment phase, medications are often prescribed to reduce the pain experience to make the patient more comfortable. Frequently, conventional over-the-counter analgesics, such as

However, opioid analgesics such as hydrocodone/paracetamol (paracetamol) or hydrocodone/acetaminophen (e.g.,

) The administration of morphine (e.g.,

or

) An antioxidant, an oxycodone (e.g.,

or

) Oxymorphone hydrochloride

And fentanyl (e.g.,

) It can also be used for moderate or severe pain.

In an effort to protect normal cells from therapeutic toxicity and limit organ toxicity, cytoprotective agents (such as neuroprotective agents, free radical scavengers, cardioprotective agents (cardioprotectors), anthracycline spill neutralizers, nutritional agents, and the like) may be used as adjunctive therapies. Suitable cell protective agents include amifostine

Glutamine, dimesna (dimesna)

Mesna (mesna)

Dexrazoxane (

Or

) Zaliloden

And leucovorin (also known as leucovorin (leucovorin), nervophilia factor and leucovorin).

The structures of the active compounds identified by codes, common names or trademarks can be taken from the actual edition of the standard summary "merck index" or from databases, for example from international patents (for example IMS World Publications).

The above-mentioned compounds which may be used in combination with the compounds of the present invention may be prepared and administered as described in the art (as in the above-cited documents).

In one embodiment, the present invention provides a pharmaceutical composition comprising at least one compound of the present invention (e.g., a compound of the present invention) or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier suitable for administration to a human or animal subject, alone or in combination with other anti-cancer agents.

In one embodiment, the invention provides a method of treating a human or animal subject suffering from a cellular proliferative disease, such as cancer. The present invention provides a method of treating a human or animal subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of a compound of the invention (e.g., a compound of the invention) or a pharmaceutically acceptable salt thereof, alone or in combination with other anti-cancer agents.

In particular, the compositions will be formulated for administration together or separately as a combination therapy.

In combination therapy, the compound of the present invention and the other anticancer agent may be administered simultaneously, simultaneously or sequentially without specific time constraints, wherein such administration provides therapeutically effective levels of both compounds within the body of the patient.

In a preferred embodiment, the compound of the invention and the other anticancer agent are administered sequentially, typically by infusion or orally, in any order. The dosage regimen may depend on the stage of the disease, the physical health of the patient, the safety profile and tolerability of the individual drugs, and other criteria well known to the attending physician and medical practitioner administering the combination. The compounds of the present invention and other anticancer agents may be administered several minutes, hours, days or even weeks apart from each other, depending on the particular cycle being treated. In addition, the cycle may include administering one drug more often than another during the treatment cycle and at different doses each time a drug is administered.

In another aspect of the invention, kits are provided comprising one or more compounds of the invention and a combination partner as disclosed herein. Representative kits comprise (a) a compound of the invention or a pharmaceutically acceptable salt thereof, (b) at least one combination partner, e.g., as indicated above, such kits thus may contain a package insert or other indication, including instructions for administration.

The compounds of the invention can also be used in combination with known therapeutic methods, for example to facilitate the administration of hormones or, in particular, radiotherapy. The compounds of the invention are particularly useful as radiosensitizers, particularly for the treatment of tumors that show poor sensitivity to radiation therapy.

In one embodiment, a drug that reduces or ameliorates side effects associated with administration of a CAR-expressing cell can be administered to a subject. Side effects associated with administration of cells expressing the CAR include, but are not limited to, CRS and lymphohistiocytosis haemophilus (HLH), also known as Macrophage Activation Syndrome (MAS). Symptoms of CRS include high fever, nausea, transient hypotension, hypoxia, and the like. CRS may include clinical signs and symptoms such as fever, fatigue, anorexia, myalgia, arthralgia, nausea, vomiting, and headache. CRS may include clinical skin signs and symptoms such as skin rash. CRS may include gastrointestinal signs and symptoms such as nausea, vomiting, and diarrhea. CRS may include respiratory clinical signs and symptoms such as tachypnea and hypoxemia. CRS may include cardiovascular clinical signs and symptoms such as tachycardia, widened pulse pressure, hypotension, increased cardiac output (early stage) and possibly impaired cardiac output (late stage). CRS may include clinical signs and symptoms of coagulation, such as elevated d-dimer, hypofibrinogenemia, with or without bleeding. CRS may include renal clinical signs and symptoms such as azotemia. CRS may include liver clinical signs and symptoms such as elevated transaminases and hyperbilirubinemia. CRS may include neurological clinical signs and symptoms such as headache, altered mental state, confusion, delirium, difficulty finding words or apparent aphasia, hallucinations, tremor, dysdiscrimination, altered gait, and seizures.

Thus, the methods described herein can include administering a CAR-expressing cell described herein to a subject and further administering one or more drugs to manage the elevated level of soluble factors resulting from the CAR-expressing cell therapy. In one embodiment, the elevated soluble factor in the subject is one or more of IFN- γ, TNF α, IL-2, and IL-6. In one embodiment, the elevated factor in the subject is one or more of IL-1, GM-CSF, IL-10, IL-8, IL-5, and fraktalk. Thus, the drug administered to treat this side effect may be one that neutralizes one or more of these soluble factors. In one embodiment, the agent that neutralizes one or more of these soluble forms is an antibody or antibody fragment. Examples of such drugs include, but are not limited to, steroids (e.g., corticosteroids), inhibitors of TNF α, and inhibitors of IL-6. Examples of TNF α inhibitors are anti-TNF α antibody molecules, such as infliximab (infliximab), adalimumab (adalimumab), certolizumab pegol (certolizumab pegol), and golimumab (golimumab). Another example of a TNF α inhibitor is a fusion protein such as entanercept. Small molecule inhibitors of TNF α include, but are not limited to, xanthine derivatives (e.g., pentoxifylline) and bupropion. Examples of IL-6 inhibitors are anti-IL-6 antibody molecules such as toclizumab (toc), sarilumab, elsilimomab, CNTO328, ALD518/BMS-945429, CNTO136, CPSI-2364, CDP6038, VX30, ARGX-109, FE301, and FM 101. In one embodiment, the anti-IL-6 antibody molecule is tollizumab. An example of an inhibitor based on IL-1R is anakinra.

In some embodiments, a corticosteroid, such as, for example, methylprednisolone, hydrocortisone, and the like, is administered to the subject.

In some embodiments, a vasopressor agent, such as, for example, norepinephrine, dopamine, phenylephrine, epinephrine, vasopressin, or a combination thereof, is administered to the subject.

In one embodiment, an antipyretic may be administered to a subject. In one embodiment, an analgesic may be administered to the subject.

In one embodiment, the subject can be administered an agent that enhances the activity of the CAR-expressing cell. For example, in one embodiment, the drug may be a drug that inhibits an inhibitory molecule, e.g., the drug is a checkpoint inhibitor. In some embodiments, an inhibitory molecule (e.g., programmed death 1(PD1)) can reduce the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR β. Inhibition of the inhibitory molecule (e.g., by inhibition at the DNA, RNA, or protein level) can optimize the performance of the cell expressing the CAR. In embodiments, for example, as described herein, an inhibitory nucleic acid (e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA), a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), a transcription activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN) can be used to inhibit expression of an inhibitory molecule in a cell expressing a CAR. In one embodiment, the inhibitor is an shRNA. In one embodiment, an inhibitory molecule is inhibited inside a cell expressing the CAR. In these embodiments, a dsRNA molecule that inhibits expression of an inhibitory molecule is linked to a nucleic acid encoding a component (e.g., all components) of the CAR.

In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits expression of a molecule that modulates or regulates (e.g., inhibits) T cell function is operably linked to a promoter, e.g., H1-or U6-derived promoter, thereby inhibiting expression of the dsRNA molecule that modulates or regulates (e.g., inhibits) expression of the molecule that modulates or regulates (e.g., inhibits) T cell function, e.g., is expressed inside a cell that expresses the CAR. See, e.g., Tiscornia G., "Development of Lentiviral Vectors Expressing siRNA," Chapter 3, from Gene Transfer: Delivery and Expression of DNA and RNA (editors Friedmann and Ross.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007; brummelkamp TR et al, (2002) Science 296: 550-; miyagishi M et al, (2002) nat. Biotechnol.19: 497-Asn 500. In one embodiment, the nucleic acid molecule encoding the dsRNA molecule that inhibits expression of a molecule that modulates or regulates (e.g., inhibits) T cell function is present on the same vector (e.g., a lentiviral vector) that comprises the nucleic acid molecule encoding the CAR component (e.g., all of its components). In such embodiments, the nucleic acid molecule encoding the dsRNA molecule that inhibits expression of a molecule that modulates or regulates (e.g., inhibits) T cell function is located on a vector (e.g., a lentiviral vector) 5 'or 3' relative to the nucleic acid encoding the CAR component (e.g., all of its components). A nucleic acid molecule encoding a dsRNA molecule that inhibits expression of a molecule that modulates or regulates (e.g., inhibits) T cell function can be transcribed in the same or different directions as the nucleic acid encoding the CAR component (e.g., all of its components). In one embodiment, the nucleic acid molecule encoding the dsRNA molecule that inhibits expression of a molecule that modulates or regulates (e.g., inhibits) T cell function is present on a vector other than a vector comprising the nucleic acid molecule encoding the CAR component (e.g., all of its components). In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits expression of a molecule that modulates or regulates (e.g., inhibits) T cell function is transiently expressed within a cell that expresses the CAR. In one embodiment, the nucleic acid molecule encoding the dsRNA molecule that inhibits expression of a molecule that modulates or regulates (e.g., inhibits) T cell function is stably integrated into the genome of the CAR-expressing cell. Figures 52A-52E depict examples of vectors that express a CAR component (e.g., all of its components) and a dsRNA molecule that inhibits expression of a molecule that modulates or modulates (e.g., inhibits) T cell function.

Examples of dsRNA molecules that can be used to inhibit expression of a molecule that modulates or modulates (e.g., inhibits) T cell function are provided below, wherein the molecule that modulates or modulates (e.g., inhibits) T cell function is PD-1.

The names of PDCD1(PD1) RNAi agents (derived from the position thereof in the mouse PDCD1 gene sequence NM-008798.2) are provided in Table 7 below, along with SEQ ID NO:159-206 representing the DNA sequence. Both sense (S) and Antisense (AS) sequences are presented in the table AS the 19 mer and 21 mer sequences. Also indicated is the position (PoS, e.g., 176) derived from the position number in the mouse PDCD1 gene sequence NM _ 008798.2. The SEQ ID NOS are shown in 12 groups corresponding to the "sense 19" SEQ ID NO: 159-170; "sense 21" SEQ ID NO 171-182; "antisense 21" SEQ ID NO 183-194; "antisense 19" SEQ ID NO: 195-206.

TABLE 7 mouse PDCD1(PD1) shRNA sequences

The names of PDCD1(PD1) RNAi agents (derived from their position in the human PDCD1 gene sequence) are provided in Table 8 below, along with SEQ ID NO:207-254, which represents the DNA sequence. Both the sense (S) and Antisense (AS) sequences are presented AS the 19-mer and 21-mer sequences. The SEQ ID NOS are shown in 12 groups corresponding to the "sense 19" SEQ ID NO: 207-218; "sense 21" SEQ ID NO: 219-230; "antisense 21" SEQ ID NO 231-242; "antisense 19" SEQ ID NO 243-254.

TABLE 8 human PDCD1(PD1) shRNA sequences

In one embodiment, the inhibitor of the inhibitory signal may be, for example, an antibody or antibody fragment that binds to the inhibitory molecule. For example, the inhibitor can be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2, or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101 and as

Selling; Bristol-Myers Squibb; tremelimumab (tremelimumab) (the IgG2 monoclonal antibody available from Pfizer, previously known as ticilimumab, CP-675206)). In one embodiment, the inhibitor is an antibody or antibody fragment that binds to TIM 3. In one embodiment, the inhibitor is an antibody or antibody fragment that binds to LAG 3. In embodiments, the agent that enhances the activity of the CAR-expressing cell (e.g., an inhibitor of an inhibitory molecule) is administered in combination with an allogeneic CAR (e.g., an allogeneic CAR described herein (e.g., described in the "allogeneic CAR" section herein)).

PD1 is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD1 is expressed on activated B cells, T cells and myeloid cells (Agata et al, 1996int. Immunol 8: 765-75). Two ligands of PD1, PD-L1 and PD-L2, have been shown to down-regulate T cell activation when bound to PD1 (Freeman et al, 2000J Exp Med 192: 1027-34; Latchman et al, 2001 Nat Immunol 2: 261-8; Carter et al, 2002Eur J Immunol 32: 634-43). PD-L1 is abundant in human cancers (Dong et al, 2003J Mol Med 81: 281-7; Blank et al, 2005 Cancer Immunol. Immunother 54: 307-314; Konishi et al, 2004 Clin Cancer Res 10: 5094). Immunosuppression can be reversed by inhibiting the local interaction of PD1 with PD-L1. Antibodies, antibody fragments, and other inhibitors of PD1, PD-L1, and PD-L2 are available in the art and can be used in combination with the CD33 CARs described herein. For example, nivolumab (also known as BMS-936558 or MDX 1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody that specifically blocks PD 1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in US 8,008,449 and WO 2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1Pidilizumab and another humanized anti-PD 1 monoclonal antibody disclosed in WO 2009/101611. Lammbrolizumab (also known as MK 03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD 1. Lambrolizumab and other humanized anti-PD 1 antibodies are disclosed in US 8,354,509 and WO 2009/114335. MDPL3280A (Genentech/Roche) is a human Fc-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies against PD-L1 are disclosed in U.S. patent No. 7,943,743 and U.S. publication No. 20120039906. Other anti-PD-L1 binders include yw243.55.s70 (heavy and light chain variable regions are shown in SEQ ID NOs: 20 and 21 of WO 2010/077634) and MDX-1105 (also known as BMS-936559 and an anti-PD-L1 binder as disclosed in, for example, WO 2007/005874). AMP-224 (B7-DCIg; Amplimmune; e.g., as disclosed in WO 2010/027827 and WO 2011/066342) is a PD-L2Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1. Other anti-PD-1 antibodies include AMP514 (amplimune) and the like, e.g., anti-PD 1 antibodies disclosed in US 8,609,089, US 2010028330, and/or US 20120114649.

TIM3(T cell immunoglobulin-3) also negatively regulates T cell function, particularly in IFN-g-secreting CD4+ helper T cell 1 and CD8+ T cytotoxic 1 cells, and plays a crucial role in T cell depletion. Inhibition of the interaction between TIM3 and its ligands (e.g., galectin-9 (Gal9), Phosphatidylserine (PS), and HMGB1) may increase the immune response. Antibodies, antibody fragments, and other inhibitors of TIM3 and its ligands are available in the art and can be used in combination with the CD19 CARs described herein. For example, antibodies, antibody fragments, small molecules, or peptide inhibitors that target TIM3 bind to the IgV domain of TIM3 to inhibit interaction with its ligands. Antibodies and peptides that inhibit TIM3 are disclosed in WO 2013/006490 and US 20100247521. Other anti-TIM 3 antibodies include humanized versions of RMT3-23 (Ngiow et al, 2011, disclosed in Cancer Res,71: 3540-. Bispecific antibodies that inhibit TIM3 and PD-1 are disclosed in US 20130156774.

In other embodiments, the agent that enhances the activity of the CAR-expressing cell is a CEACAM inhibitor (e.g., a CEACAM-1, CEACAM-3, and/or CEACAM-5 inhibitor). In one embodiment, the inhibitor of CEACAM is an anti-CEACAM antibody molecule. Exemplary anti-CEACAM-1 antibodies are described in WO 2010/125571, WO 2013/082366, WO 2014/059251, and WO 2014/022332, e.g., monoclonal antibodies 34B1, 26H7, and 5F 4; or a recombinant form thereof, e.g. as described in US 2004/0047858, US7,132,255 and WO 99/052552. In other embodiments, the anti-CEACAM antibody binds CEACAM-5, e.g., Zheng et al, PLoS one.2010 Sep 2; pii: e12529(DOI:10:1371/journal. pane.0021146), or cross-reacting with CEACAM-1 and CEACAM-5, for example as described in WO 2013/054331 and US 2014/0271618.

Without wishing to be bound by theory, it is believed that carcinoembryonic antigen cell adhesion molecules (CEACAM), such as CEACAM-1 and CEACAM-5, mediate, at least in part, inhibition of immune responses against tumors (see, e.g., Markel et al, J Immunol.2002 Mar 15; 168(6): 2803-10; Markel et al, J Immunol.2006Nov 1; 177(9): 6062-71; Markel et al, Immunology.2009 Feb; 126(2): 186-200; Markel et al, Cancer Immunol.2010 Feb; 59(2): 215-30; Ortenberg et al, Mol Cancer The 2012 Jun; 11(6): 1300-10; Ste et al, J Unol 2005.JJ.174; heng 92; Zosi. 2010; Sep. 2010; Zhao et al; Sep.1259; Sep.29). For example, CEACAM-1 has been described as an heterophilic ligand of TIM-3 and plays a role in TIM-3 mediated T cell tolerance and depletion (see, e.g., WO 2014/022332; Huang et al, (2014) Nature doi:10.1038/Nature 13848). In embodiments, co-blocking CEACAM-1 and TIM-3 has been shown to enhance anti-tumor immune responses in xenograft colorectal cancer models (see, e.g., WO 2014/022332; Huang et al, (2014), supra). In other embodiments, co-blocking CEACAM-1 and PD-1 reduces T cell tolerance, e.g., as described in WO 2014/059251. Thus, CEACAM inhibitors may be used with other immunomodulatory agents described herein (e.g., anti-PD-1 and/or anti-TIM-3 inhibitors) to enhance immune responses against cancers (e.g., melanoma, lung cancer (e.g., NSCLC), bladder cancer, colon cancer, ovarian cancer, and other cancers as described herein).

LAG3 (lymphocyte activation gene-3 or CD223) is a cell surface molecule expressed on activated T and B cells, which has been shown to play a role in CD8+ T cell depletion. Antibodies, antibody fragments, and other inhibitors of LAG3 and its ligands are available in the art and may be used in combination with the CD19 CARs described herein. For example, BMS-986016(Bristol-Myers Squib) is a monoclonal antibody targeting LAG 3. IMP701(Immutep) is the antagonist LAG3 antibody and IMP731(Immutep and GlaxoSmithKline) is the depleting LAG3 antibody. Other LAG3 inhibitors include IMP321(Immutep), which IMP321 is a recombinant fusion protein of the soluble portion of LAG3 and Ig that binds to MHC class II molecules and activates Antigen Presenting Cells (APCs). Other antibodies are disclosed, for example, in WO 2010/019570.

In some embodiments, the agent that enhances the activity of a cell expressing the CAR can be, for example, a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule or fragment thereof and the second domain is a polypeptide associated with a forward signal, e.g., a polypeptide comprising an intracellular signaling domain as described herein. In some embodiments, the polypeptide associated with a forward signal may comprise a co-stimulatory domain of CD28, CD27, ICOS, e.g., the intracellular signaling domain of CD28, CD27, and/or ICOS and/or the primary signaling domain (e.g., of CD3 ζ) as described herein. In one embodiment, the fusion protein is expressed by the same cell that expresses the CAR. In another embodiment, the fusion protein is expressed by a cell (e.g., a T cell) that does not express a CD33 CAR.

In one embodiment, the agent that enhances the activity of a CAR-expressing cell described herein is miR-17-92.

In one embodiment, the agent that enhances the activity of a CAR-expressing cell described herein is a cytokine. Cytokines have important functions related to T cell expansion, differentiation, survival and homeostasis. Cytokines that can be administered to a subject receiving a CAR-expressing cell described herein include: IL-2, IL-4, IL-7, IL-9, IL-15, IL-18 and IL-21 or combinations thereof. In a preferred embodiment, the cytokine administered is IL-7, IL-15, or IL-21, or a combination thereof. The cytokine may be administered once a day or more than once a day, e.g., 2 times a day, 3 times a day, or 4 times a day. The cytokine may be administered for more than one day, e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. For example, the cytokine is administered once a day for 7 days.

In embodiments, the cytokine is administered in combination with a CAR-expressing T cell. The cytokine can be administered simultaneously or concurrently with the CAR-expressing T cells, e.g., on the same day. The cytokine may be formulated in the same pharmaceutical composition as the CAR-expressing T cell, or may be formulated in an isolated pharmaceutical composition. Alternatively, the cytokine may be administered shortly after administration of the CAR-expressing T-cell, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the CAR-expressing T-cell. In embodiments where the cytokine is administered in a dosing regimen that is performed for more than one day, day 1 of the cytokine dosing regimen may be on the same day as administration of the CAR-expressing T cells, or day 1 of the cytokine dosing regimen may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the CAR-expressing T cells. In one embodiment, on day 1, CAR-expressing T cells are administered to the subject and on day 2, cytokines are administered once a day for the next 7 days. In a preferred embodiment, the cytokine administered in combination with the CAR-expressing T cells is IL-7, IL-15 or IL-21.

In other embodiments, the cytokine is administered at a time after administration of the CAR-expressing cells, e.g., at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more after administration of the CAR-expressing cells. In one embodiment, the cytokine is administered after assessing the subject's response to the CAR-expressing cell. For example, the CAR-expressing cells are administered to the subject according to the dosages and treatment regimens described herein. Assessing a subject's response to a CAR-expressing cell therapy, including inhibition of tumor growth, reduction of circulating tumor cells, or tumor regression, using any of the methods described herein at 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more after administration of the CAR-expressing cells. The cytokine can be administered to a subject that does not show a sufficient response to the CAR-expressing cell therapy. Administration of cytokines to subjects who are suboptimal for CAR-expressing cell therapy improves the efficacy or anti-cancer activity of the CAR-expressing cells. In a preferred embodiment, the cytokine administered after administration of the CAR-expressing cells is IL-7.

Combination with immunopotentiating low doses of mTOR inhibitors

The methods described herein use immunopotentiating low doses of mTOR inhibitors, e.g., allosteric mTOR inhibitors, including rapamycin analogs such as RAD 001. Administration of an immunopotentiating low dose of an mTOR inhibitor (e.g., a dose that is insufficient to completely suppress the immune system, but sufficient to improve immune function) can optimize the performance of immune effector cells (e.g., T cells or CAR-expressing cells) in a subject. Methods, dosages, treatment regimens and suitable pharmaceutical compositions for measuring mTOR inhibition are described in U.S. patent application No. 2015/01240036, which is hereby incorporated by reference.

In one embodiment, administration of an immunopotentiating low dose of an mTOR inhibitor may result in one or more of the following:

i) a decrease in the number of PD-1 positive immune effector cells;

ii) an increase in the number of PD-1 negative immune effector cells;

iii) an increased ratio of PD-1 negative immune effector cells/PD-1 positive immune effector cells;

iv) an increase in the number of naive T cells;

v) increased expression of one or more of the following markers: for example, CD62L on memory T cells (e.g., memory T cell precursors) ^{Height of}、CD127^{Height of}、CD27⁺And BCL 2;

vi) reduced expression of KLRG1 on memory T cells (e.g., memory T cell precursors); or

vii) an increase in the number of memory T cell precursors, such as cells having any one or combination of the following characteristics: CD62L^{Height of}Increase, CD127^{Height of}Increase, CD27⁺Increase, decrease KLRG1, and increase BCL 2;

and wherein, for example, a change in any of the foregoing, e.g., i), ii), iii), iv), v), vi), or vii), occurs, e.g., at least temporarily, as compared to an untreated subject.

In another embodiment, for example, administration of an immunopotentiating low dose of an mTOR inhibitor results in increased or prolonged proliferation or persistence (e.g., in culture or in a subject) of CAR-expressing cells as compared to untreated CAR-expressing cells or untreated subjects. In embodiments, the increased proliferation or persistence is associated with an increase in the number of cells expressing the CAR. Methods for measuring increased or prolonged proliferation are described in examples 8 and 9. In another embodiment, for example, administration of an immunopotentiating low dose of an mTOR inhibitor results in (e.g., in culture or in a subject) increased cancer cell killing of CAR-expressing cells as compared to untreated CAR-expressing cells or untreated subjects. In embodiments, the increased killing of cancer cells is associated with a reduction in tumor volume. A method for measuring increased cancer cell killing is described in example 6.

In one embodiment, a cell expressing a CAR molecule (e.g., a CAR molecule described herein) is administered in combination with an immunopotentiating low dose of an mTOR inhibitor (e.g., an allosteric mTOR inhibitor, e.g., RAD001 or a catalytic mTOR inhibitor). For example, administration of an immunopotentiating low dose of an mTOR inhibitor may be initiated prior to administration of the CAR-expressing cells described herein; is done prior to administration of the CAR-expressing cells described herein; is initiated simultaneously with administration of a CAR-expressing cell described herein; overlapping with administration of a CAR-expressing cell described herein; or continued after administration of the CAR-expressing cells described herein.

Alternatively or additionally, administration of an immunopotentiating low dose of an mTOR inhibitor may optimize immune effector cells to be engineered to express the CAR molecules described herein. In such embodiments, administration of an immunopotentiating low dose of an mTOR inhibitor (e.g., an allosteric inhibitor (e.g., RAD001) or a catalytic inhibitor) is initiated or completed prior to harvesting from the subject an immune effector cell (e.g., a T cell or NK cell)) to be engineered to express a CAR molecule described herein.

In another embodiment, the immune effector cells (e.g., T cells or NK cells) to be engineered to express a CAR molecule described herein, e.g., after harvesting from a subject, or the immune effector cells (e.g., T cells or NK cells) expressing a CAR, e.g., prior to administration to a subject, can be cultured in the presence of an immunopotentiating low dose of an mTOR inhibitor.

In one embodiment, administering to the subject an immunopotentiating low dose of an mTOR inhibitor includes, for example, administering 0.1 to 20, 0.5 to 10, 2.5 to 7.5, 3 to 6, or about 5mg RAD001, or a bioequivalent dose thereof, once per week, e.g., in an immediate release dosage form. In one embodiment, administering to the subject an immunopotentiating low dose of an mTOR inhibitor includes, e.g., once per week, e.g., administering 0.3 to 60, 1.5 to 30, 7.5 to 22.5, 9 to 18, or about 15mg RAD001, or a bioequivalent dose thereof, in a sustained release dosage form.

In one embodiment, the dosage of the mTOR inhibitor is commensurate with at least 5% but not more than 90%, at least 10% but not more than 90%, at least 15% but not more than 90%, at least 20% but not more than 90%, at least 30% but not more than 90%, at least 40% but not more than 90%, at least 50% but not more than 90%, at least 60% but not more than 90%, at least 70% but not more than 90%, at least 5% but not more than 80%, at least 10% but not more than 80%, at least 15% but not more than 80%, at least 20% but not more than 80%, at least 30% but not more than 80%, at least 40% but not more than 80%, at least 50% but not more than 80%, at least 60% but not more than 80%, at least 5% but not more than 70%, at least 10% but not more than 70%, at, At least 20% but not more than 70%, at least 30% but not more than 70%, at least 40% but not more than 70%, at least 50% but not more than 70%, at least 5% but not more than 60%, at least 10% but not more than 60%, at least 15% but not more than 60%, at least 20% but not more than 60%, at least 30% but not more than 60%, at least 40% but not more than 60%, at least 5% but not more than 50%, at least 10% but not more than 50%, at least 15% but not more than 50%, at least 20% but not more than 50%, at least 30% but not more than 50%, at least 40% but not more than 50%, at least 5% but not more than 40%, at least 10% but not more than 40%, at least 15% but not more than 40%, at least 20% but not more than 40%, at least 30% but not more, At least 5% but not more than 30%, at least 10% but not more than 30%, at least 15% but not more than 30%, at least 20% but not more than 30%, or at least 25% but not more than 30% of mTOR inhibition is associated with or provides such mTOR inhibition.

The degree of mTOR inhibition may be expressed as or correspond to the degree of P70S6 kinase inhibition, for example the degree of mTOR inhibition may be determined by the level of decreased P70S6 kinase activity (e.g. by decreased phosphorylation of the P70S6 kinase substrate). The level of mTOR inhibition can be assessed by a variety of methods, such as measuring P70S6 kinase activity by the Boulay assay, as described in U.S. patent application No. 2015/01240036, which is hereby incorporated by reference, or as described in U.S. patent No. 7,727,950, which is hereby incorporated by reference; measuring the level of phosphorylated S6 by western blotting; or to evaluate changes in the ratio of PD 1-negative versus PD 1-positive immune effector cells.

As used herein, the term "mTOR inhibitor" refers to a compound or ligand, or a pharmaceutically acceptable salt thereof, that inhibits mTOR kinase in a cell. In one embodiment, the mTOR inhibitor is an allosteric inhibitor. Allosteric mTOR inhibitors include the neutral tricyclic compound rapamycin (sirolimus), rapamycin related compounds that are compounds having structural and functional similarities to rapamycin, including, for example, rapamycin derivatives, rapamycin analogs (also known as rapamycin analogs), and other macrocyclic lactones that inhibit mTOR activity. In one embodiment, the mTOR inhibitor is a catalytic inhibitor.

Rapamycin is a known macrolide antibiotic produced by Streptomyces hygroscopicus (Streptomyces hygroscopicus) and has the structure shown in formula a.

See, e.g., McAlpine, j.b., et al, j.antibiotics (1991)44: 688; schreiber, s.l. et al, j.am.chem.soc. (1991)113: 7433; U.S. Pat. No. 3,929,992. There are a number of numbering schemes proposed for rapamycin. To avoid confusion, when a particular rapamycin analog is named herein, the name is given with reference to rapamycin using the numbering scheme of formula a.

Rapamycin analogues useful in the present invention are, for example, O-substituted analogues in which the hydroxy group on the cyclohexyl ring of rapamycin is replaced by a group in which R is₁OR being hydroxyalkyl, hydroxyalkoxyalkyl, amidoalkyl OR aminoalkyl₁Replacement; for example, RAD001, also known as everolimus, as described in US 5,665,772 and WO 94/09010, the contents of each of which are incorporated by reference.

Other suitable rapamycin analogues include those substituted at the 26-or 28-position. The rapamycin analogue may be an epimer of the analogue mentioned above, in particular an epimer of the analogue substituted at

position

40, 28 or 26, and may optionally be further hydrogenated, for example as described in US6,015,815, WO 95/14023 and WO 99/15530, the contents of which are incorporated by reference, for example ABT578, also known as zotasol or the rapamycin analogue described in US 7,091,213, WO 98/02441 and WO 01/14387, the contents of which are incorporated by reference, for example AP23573, also known as diphospholimus.

Examples of rapamycin analogues suitable for use in the present invention from U.S. Pat. No. 5,665,772 include, but are not limited to, 40-O-benzyl-rapamycin, 40-O- (4 ' -hydroxymethyl) benzyl-rapamycin, 40-O- [4 ' - (1, 2-dihydroxyethyl) ] benzyl-rapamycin, 40-O-allyl-rapamycin, 40-O- [3 ' - (2, 2-dimethyl-1, 3-dioxolan-4 (S) -yl) -prop-2 ' -en-1 ' -yl ] -rapamycin, (2 ' E,4 ' S) -40-O- (4 ', 5 ' -dihydroxypent-2 ' -en-1 ' -yl) -rapamycin, and the like, 40-O- (2-hydroxy) ethoxycarbonylmethyl-rapamycin, 40-O- (2-hydroxy) ethyl-rapamycin, 40-O- (3-hydroxy) propyl-rapamycin, 40-O- (6-hydroxy) hexyl-rapamycin, 40-O- [2- (2-hydroxy) ethoxy ] ethyl-rapamycin, 40-O- [ (3S) -2, 2-dimethyldioxolan-3-yl ] methyl-rapamycin, 40-O- [ (2S) -2, 3-dihydroxypropan-1-yl ] -rapamycin, 40-O- (2-acetoxy) ethyl-rapamycin, 40-O- (2-nicotinic-yloxy) ethyl-rapamycin, rapamycin, 40-O- [2- (N-morpholino) acetoxy ] ethyl-rapamycin, 40-O- (2-N-imidazolylacetoxy) ethyl-rapamycin, 40-O- [2- (N-methyl-N' -piperazinyl) acetoxy ] ethyl-rapamycin, 39-O-desmethyl-39, 40-O, O-ethylene-rapamycin, (26R) -26-dihydro-40-O- (2-hydroxy) ethyl-rapamycin, 40-O- (2-aminoethyl) -rapamycin, 40-O- (2-acetamido) carbamic acid) -rapamycin, 40-O- (2-nicoylaminoethyl) carbamic acid) -rapamycin, and mixtures thereof, 40-O- (2- (N-methylimidazol-2 ' -ylethoxycarbonylamino) ethyl) -rapamycin, 40-O- (2-ethoxycarbonylaminoethyl) -rapamycin, 40-O- (2-tolylsulfonamidoethyl) -rapamycin and 40-O- [2- (4 ', 5 ' -dicarboethoxy-1 ', 2 ', 3 ' -triazol-1 ' -yl) -ethyl ] -rapamycin.

Other rapamycin analogues useful in the present invention are those in which the hydroxyl group on the cyclohexyl ring of rapamycin and/or the hydroxyl group at position 28 is replaced with a hydroxyl ester group, for example, the rapamycin analogues found in US RE44,768, such as temsirolimus.

Other rapamycin analogues that may be used in the foregoing invention include those in which the methoxy group at position 16 is replaced with another substituent, preferably an (optionally hydroxy-substituted) alkynyloxy, benzyl, o-methoxybenzyl or chlorobenzyl group and/or in which the methoxy group at position 39 is deleted along with the 39 carbon so that the cyclohexyl ring of rapamycin becomes a cyclopentyl ring which lacks the methoxy group at position 39; for example, as described in WO 95/16691 and WO96/41807, the contents of which are incorporated herein by reference. The analogs may be further modified so that the hydroxyl group at the 40 position of rapamycin is alkylated and/or the 32-carbonyl group is reduced.

Rapamycin analogues from WO 95/16691 include, but are not limited to, 16-desmethoxy-16- (pent-2-ynyl) oxy-rapamycin, 16-desmethoxy-16- (but-2-ynyl) oxy-rapamycin, 16-desmethoxy-16- (propargyl) oxy-rapamycin, 16-desmethoxy-16- (4-hydroxy-but-2-ynyl) oxy-rapamycin, 16-desmethoxy-16-benzyloxy-40-O- (2-hydroxyethyl) -rapamycin, 16-desmethoxy-16-benzyloxy-rapamycin, 16-desmethoxy-16-O-methoxybenzyl-rapamycin, 16-desmethoxy-40-O- (2-methoxyethyl) -16-pent-2-ynyl) oxy-rapamycin, 39-desmethoxy-40-deoxo-39-formyl-42-desmethyl-rapamycin, 39-desmethoxy-40-deoxo-39-hydroxymethyl-42-desmethyl-rapamycin, 39-desmethoxy-40-deoxo-39-carboxy-42-desmethyl-rapamycin, 39-desmethoxy-40-deoxo-39- (4-methyl-piperazin-1-yl) carbonyl-42-desmethyl-rapamycin, 39-desmethoxy-40-deoxo-39- (morpholin-4-yl) carbonyl -42-nor-rapamycin, 39-desmethoxy-40-deoxo-39- [ N-methyl, N- (2-pyridin-2-yl-ethyl) ] carbamoyl-42-nor-rapamycin and 39-desmethoxy-40-deoxo-39- (p-toluenesulfonylhydrazone methyl) -42-nor-rapamycin.

Rapamycin analogues from WO 96/41807 include, but are not limited to, 32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-40-O- (2-hydroxy-ethyl) -rapamycin, 16-O-pent-2-ynyl-32- (S) -dihydro-40-O- (2-hydroxyethyl) -rapamycin, 32(S) -dihydro-40-O- (2-methoxy) ethyl-rapamycin and 32(S) -dihydro-40-O- (2-hydroxyethyl) -rapamycin.

Another suitable rapamycin analog is umirolimus (umirolimus) as described in US 2005/0101624, the contents of which are incorporated by reference.

RAD001 otherwise known as everolimus

Having the chemical name (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R) -1, 18-dihydroxy-12- { (1R) -2- [ (1S,3R,4R) -4- (2-hydroxyethoxy) -3-methoxycyclohexyl]-1-methylethyl } -19, 30-dimethoxy-15, 17,21,23,29, 35-hexamethyl-11, 36-dioxa-4-aza-tricyclo [30.3.1.04,9]Thirty-seven-carbon-16, 24,26, 28-tetraene-2, 3,10,14, 20-pentaketone, as described in US 5,665,772 and WO 94/09010, the contents of each of which are incorporated by reference.

Other examples of allosteric mTOR inhibitors include sirolimus (rapamycin, AY-22989), 40- [ 3-hydroxy-2- (hydroxymethyl) -2-methylpropionate ] -rapamycin (also known as temsirolimus or CCI-779), and diphospholimus (AP-23573/MK-8669). Other examples of allosteric mTOR inhibitors include zotarolimus (ABT578) and umirolimus.

Alternatively or additionally, ATP-competitive catalytic mTOR inhibitors have been found to directly target the mTOR kinase domain and to target both mTORC1 and mTORC 2. There are also mTORC1 inhibitors that are more potent than such allosteric mTOR inhibitors such as rapamycin, as they modulate rapamycin-resistant mTORC1 output, such as 4EBP1-T37/46 phosphorylation and cap-dependent translation.

Catalytic inhibitors include: BEZ235 or 2-methyl-2- [4- (3-methyl-2-oxo-8-quinolin-3-yl-2, 3-dihydro-imidazo [4,5-c ]]Quinolin-1-yl) -phenyl]-propionitrile or the monotosylate salt form (synthesis of BEZ235 is described in WO 2006/122806; CCG168 (otherwise known as AZD-8055, Chresta, C.M. et al, Cancer Res,2010,70(1),288-298) having the chemical name {5- [2, 4-bis- ((S) -3-methyl-morpholin-4-yl) -pyrido [2,3d ] -pyridino- [2, 4-methyl-morpholin-4-yl ]]Pyrimidin-7-yl]-2-methoxy-phenyl } -methanol; 3- [2, 4-bis [ (3S) -3-methylmorpholin-4-yl]Pyrido [2,3-d]Pyrimidin-7-yl]-N-methylbenzamide (WO 09104019); 3- (2-aminobenzo [ d ]]

Azol-5-yl) -1-isopropyl-1H-pyrazolo [3,4-d]Pyrimidin-4-amines (WO 10051043 and WO 2013023184); a N- (3- (N- (3- ((3, 5-dimethoxyphenyl) amino) quinoline)

Lin-2-yl) sulfamoyl) phenyl) -3-methoxy-4-methylbenzamide (WO 07044729 and WO 12006552); PKI-587(Venkatesan, A.M., J.Med.chem.,2010,53,2636-2645) having the chemical name 1- [4- [4- (dimethylamino) piperidine-1-carbonyl-) ]Phenyl radical]-3- [4- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) phenyl]Urea; GSK-2126458(ACS Med. chem. Lett.,2010,1,39-43) having the chemical name 2, 4-difluoro-N- { 2-methoxy-5- [4- (4-pyridazinyl) -6-quinolinyl]-3-pyridinyl } benzenesulfonamide; 5- (9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl) pyrimidin-2-amine (WO 10114484); and (E) -N- (8- (6-amino-5- (trifluoromethyl) pyridin-3-yl) -1- (6- (2-cyanoprop-2-yl) pyridin-3-yl) -3-methyl-1H-imidazo [4,5-c]Quinolin-2 (3H) -ylidene) cyanamide (WO 12007926).

Other examples of catalytic mTOR inhibitors include 8- (6-methoxy-pyridin-3-yl) -3-methyl-1- (4-piperazin-1-yl-3-trifluoromethyl-phenyl) -1, 3-dihydro-imidazo [4,5-c ] quinolin-2-one (WO 2006/122806) and Ku-0063794(Garcia-Martinez JM et al, Biochem j.,2009,421(1), 29-42). Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR). WYE-354 is another example of a catalytic mTOR inhibitor (Yu K et al, (2009). Biochemical, Cellular, and In vivo Activity of Novel ATP-reactive and Selective Inhibitors of the Mammalian Target of rapamycin. cancer Res.69(15): 6232-6240).

mTOR inhibitors useful according to the invention also include prodrugs, derivatives, pharmaceutically acceptable salts, or analogs of any of the foregoing.

mTOR inhibitors, such as RAD001, can be formulated for delivery based on the particular dosages described herein, based on well established methods in the art. In particular, U.S. patent No. 6,004,973 (incorporated herein by reference) provides examples of formulations that are useful with the mTOR inhibitors described herein.

Methods and biomarkers for evaluating CAR effectiveness or sample suitability

In another aspect, the invention features a method of evaluating or monitoring the effectiveness of a CAR-expressing cell therapy (e.g., CD33 CAR therapy) in a subject (e.g., a subject having a cancer (e.g., a hematological cancer)) or sample (e.g., apheresis) suitability of a CAR therapy (e.g., CD33 CAR therapy). The method comprises obtaining a value for the effectiveness of the CAR therapy or the suitability of the sample, wherein the value represents the effectiveness or suitability of a cell therapy expressing the CAR.

In embodiments, the value of CAR therapy effectiveness or sample suitability comprises measuring one, two, three, four, five, six, or more (all) of:

(i) the level or activity of one, two, three, or more (e.g., all) of the following in a sample (e.g., an apheresis sample or a CAR-expressing cell product sample produced): resting type T _EFFCell, resting type T_REGCells, younger T cells (e.g., younger CD4 cells or CD8 cells, or γ/δ T cells), or early memory T cells, or combinations thereof;

(ii) the level or activity of one, two, three, or more (e.g., all) of the following in a sample (e.g., an apheresis sample or a CAR-expressing cell product sample produced): activated form T_EFFCellular, activated T_REGCells, older T cells (e.g., older CD4 cells or CD8 cells), or late memory T cells, or a combination thereof;

(iii) the level or activity of an immune cell depletion marker, e.g., one, two or more immune checkpoint inhibitors (e.g., PD-1, PD-L1, TIM-3, and/or LAG-3), in a sample (e.g., an apheresis sample or a CAR-expressing cell product sample produced). In one embodiment, the immune cell has a depletion phenotype, e.g., co-expresses at least two depletion markers, e.g., co-expresses PD-1 and TIM-3. In other embodiments, the immune cell has a depletion phenotype, e.g., co-expresses at least two depletion markers, e.g., co-expresses PD-1 and LAG-3.

(iii) The level or activity of CD27 and/or CD45RO- (e.g., CD27+ CD45RO-) immune effector cells, e.g., in a CD4+ or CD8+ T cell population, in a sample (e.g., an apheresis sample or a CAR-expressing cell product sample produced);

(v)1, 2, 3, 4, 5, 10, 20 or more biomarkers selected from CCL20, IL-17a and/or IL-6, PD-1, PD-L1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG 1: (ii) a

(vi) Cytokine levels or activity (e.g., quality of cytokine repertoire) in a CAR-expressing cell product sample (e.g., a CAR 33-expressing cell product sample); or

(vii) Transduction efficiency of CAR-expressing cells in the CAR-expressing cell product sample produced.

In some embodiments of any of the methods disclosed herein, the CAR-expressing cell therapy comprises a plurality of CAR-expressing immune effector cells (e.g., a population), e.g., a plurality of T cells or NK cells (e.g., a population), or a combination thereof. In one embodiment, the CAR-expressing cell therapy is a CD33 CAR therapy.

In some embodiments of any of the methods disclosed herein, the measurement of one or more of (i) - (vii) is obtained from an apheresis sample obtained from the subject. The apheresis sample may be evaluated prior to infusion or reinfusion.

In some embodiments of any of the methods disclosed herein, the measurement of one or more of (i) - (vii) is obtained from a sample of a CAR-expressing cell product produced (e.g., a CD33 CAR-expressing cell product sample). The CAR-expressing cell product produced can be evaluated prior to infusion or reinfusion.

In some embodiments of any of the methods disclosed herein, the subject is evaluated prior to, during, or after receiving the CAR-expressing cell therapy.

In some embodiments of any of the methods disclosed herein, the measurement of one or more of (i) - (vii) assesses one or more characteristics of gene expression, flow cytometry, or protein expression.

In some embodiments of any of the methods disclosed herein, the method further comprises identifying the subject as a responder, a non-responder, a relapser or a non-relapser based on the measurement of one or more of (i) - (vii).

In some embodiments of any of the methods disclosed herein, a responder (e.g., a complete responder) has or is identified as having a higher level or activity of one, two or more (all) of GZMK, PPF1BP2, or naive T cells, as compared to a non-responder.

In some embodiments of any of the methods disclosed herein, the non-responder has or is identified as having a higher level or activity of one, two, three, four, five, six, seven, or more (e.g., all) of IL22, IL-2RA, IL-21, IRF8, IL8, CCL17, CCL22, effector T cells, or regulatory T cells, as compared to the responder.

In one embodiment, a relapser is a patient having or identified as having increased expression levels of one or more (e.g., 2, 3, 4, or all) of the following genes as compared to a non-relapser: MIR199a1, MIR1203, uc021ovp, ITM2C, and HLA-DQB1 and/or has or is identified as having a reduced level of gene expression compared to a non-relapser (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all) of one or more of: PPIAL4D, TTTY10, TXLNG2P, MIR4650-1, KDM5D, USP9Y, PRKY, RPS4Y2, RPS4Y1, NCRNA00185, SULT1E1 and EIF1 AY.

In some embodiments of any of the methods disclosed herein, a full responder has or is identified as having a greater (e.g., statistically significantly greater) percentage of CD8+ T cells than a reference value (e.g., a non-responder percentage of CD8+ T cells).

In some embodiments of any of the methods disclosed herein, a full responder has or is identified as having a greater percentage of CD27+ CD45 RO-immune effector cells, e.g., in a CD8+ population, as compared to a reference value (e.g., the number of non-responder CD27+ CD45 RO-immune effector cells).

In some embodiments of any of the methods disclosed herein, a full or partial responder has or is identified as having a greater (e.g., statistically significantly greater) percentage of CD4+ T cells than a reference value (e.g., a non-responder percentage of CD4+ T cells).

In some embodiments of any of the methods disclosed herein, the ratio to a reference value (e.g., resting type T of non-responder)_EFFCell, resting type T_REGCell, younger T cell (e.g., younger CD4 cell or CD8 cell), or early memory T cell number), a complete responder has or is identified as having a greater percentage of one, two, three, or more (e.g., all) of: resting type T_EFFCell, resting type T_REGCells, younger T cells (e.g., younger CD4 cells or CD8 cells, or γ/δ T cells), or early memory T cells, or combinations thereof;

in some embodiments of any of the methods disclosed herein, a reference value (e.g., activated form T of a reactor)_EFFCellular, activated T_REGNumber of cells, older T cells (e.g., older CD4 cells or CD8 cells), or late memory T cells), non-responders have or are identified as having one, both, one or both,A greater percentage of three or more (e.g., all): activated form T_EFFCellular, activated T_REGCells, older T cells (e.g., older CD4 cells or CD8 cells), or late memory T cells, or a combination thereof.

In some embodiments of any of the methods disclosed herein, the non-responder has or is identified as having a greater percentage of an immune cell depletion marker, e.g., one, two or more immune checkpoint inhibitors (e.g., PD-1, PD-L1, TIM-3, and/or LAG-3). In one embodiment, the non-responder has or is identified as having a greater percentage of immune effector cells (e.g., CD4+ T cells and/or CD8+ T cells) expressing PD-1, PD-L1, or LAG-3 (e.g., CD4+ cells and/or CD8+ T cells expressing CAR) as compared to the percentage of immune effector cells expressing PD-1 or LAG-3 from the responder.

In one embodiment, the non-responders have or are identified as having a greater percentage of immune cells with a depletion phenotype (e.g., immune cells that co-express at least two depletion markers (e.g., co-express PD-1, PD-L1, and/or TIM-3)). In other embodiments, the non-responders have or are identified as having a greater percentage of immune cells with a depletion phenotype (e.g., immune cells that co-express at least two depletion markers (e.g., co-express PD-1 and LAG-3)).

In some embodiments of any of the methods disclosed herein, the non-responder has or is identified as having a greater percentage of PD-1/PD-L1+/LAG-3+ cells in a population of cells expressing the CAR (e.g., a CD33 CAR + cell population) than a responder (e.g., a complete responder) to the CAR-expressing cell therapy.

In some embodiments of any of the methods disclosed herein, the partial responder has or is identified as having a higher percentage of PD-1/PD-L1+/LAG-3+ cells in a population of cells expressing a CAR (e.g., a CD33 CAR + cell population) than the responder.

In some embodiments of any of the methods disclosed herein, the non-responder has or is identified as having co-expression of PD1/PD-L1+ CAR + depletion phenotype and LAG3 in a population of cells expressing the CAR (e.g., a CD33 CAR + cell population).

In some embodiments of any of the methods disclosed herein, the non-responder has or is identified as having a greater percentage of PD-1/PD-L1+/TIM-3+ cells in a population of cells expressing a CAR (e.g., a CD33 CAR + cell population) than the responder (e.g., a complete responder).

In some embodiments of any of the methods disclosed herein, the partial responder has or is identified as having a higher percentage of PD-1/PD-L1+/TIM-3+ cells in a population of cells expressing a CAR (e.g., a population of CD33 CAR + cells) than the responder.

In some embodiments of any of the methods disclosed herein, CD8+ CD27+ CD45RO-T cells present in the apheresis sample are positive predictors of response of the subject to CAR-expressing cell therapy (e.g., CD33 CAR therapy).

In some embodiments of any of the methods disclosed herein, a high percentage of PD1+ CAR + and LAG3+ or TIM3+ T cells in the apheresis sample is a poor prognostic predictor of subject response to CAR-expressing cell therapy (e.g., CD33 CAR therapy).

In some embodiments of any of the methods disclosed herein, a reactor (e.g., a full or partial reactor) has one, two, three, or more (or all) of the following characteristics:

(i) a greater number of CD27+ immune effector cells compared to a reference value (e.g., the number of non-responder CD27+ immune effector cells);

(ii) a greater number of CD8+ T cells compared to a reference value (e.g., the number of non-responder CD8+ T cells);

(iii) (ii) immune cells having a lower number of one or more checkpoint inhibitors (e.g., checkpoint inhibitors selected from PD-1, PD-L1, LAG-3, TIM-3, or KLRG-1, or a combination thereof) expressed as compared to a reference value (e.g., the number of non-responder immune cells expressing one or more checkpoint inhibitors); or

(iv) To reference value (e.g. non-responder's resting type T)_EFFCell, resting type T_REGCell, naive CD4 cells, unstimulated memory cells, or early memory T cell numbers) have a greater percentage of one, two, three, or more (e.g., all) of: resting type T _EFFCell, resting type T_REGCells, naive CD4 cells, unstimulated memory cells, or early memory T cells, or a combination thereof.

In some embodiments of any of the methods disclosed herein, the cytokine level or activity of (vi) is selected from one, two, three, four, five, six, seven, eight or more (e.g., all) of the cytokines CCL20/MIP3a, IL17A, IL6, GM-CSF, IFN γ, IL10, IL13, IL2, IL21, IL4, IL5, IL9, or TNF α, or a combination thereof. The cytokine may be selected from one, two, three, four or more (e.g., all) of IL-17a, CCL20, IL2, IL6, or TNF α. In one embodiment, an increased cytokine level or activity is selected from one or both of IL-17a and CCL20, indicating an increased responsiveness or decreased relapse.

In some embodiments of any of the methods disclosed herein, a transduction efficiency of 15% or greater in (vii) indicates an increase in responsiveness or a decrease in relapse.

In some embodiments of any of the methods disclosed herein, a transduction efficiency of less than 15% in (vii) indicates a decrease in responsiveness or an increase in relapse.

In embodiments, responders, non-responders, relapsers or non-relapsers identified by the methods herein may be further evaluated according to clinical criteria. For example, a full responder has or is identified as a subject with a disease (e.g., cancer) that shows a full response (e.g., complete remission) to treatment. As described herein, NCCN may be used, for example

Or Cheson et al, J Clin Oncol 17:1244(1999) and Cheson et al, "reviewed Response criterion for Malignant Lymphoma", J Clin Oncol 25:579-586(2007) (both of which are incorporated herein by reference in their entirety),a complete response was identified. A partial responder has or is identified as a subject with a disease (e.g., cancer) that shows a partial response (e.g., partial remission) to treatment. May, for example, use NCCN

Or a Cheson standard identification partial reaction as described herein. A non-responder has or is identified as having a subject with a disease (e.g., cancer) that does not show a response to treatment, e.g., the patient has a stable condition or a progressive condition. May, for example, use NCCN

Or identifying non-responders as the Cheson standard described herein.

Alternatively or in combination with the methods disclosed herein, one, two, three, four or more of the following is performed in response to the value:

e.g., administering a CAR-expressing cell therapy to a responder or non-relapser;

administering an altered administration of a cell therapy expressing a CAR;

altering the regimen or time course of CAR-expressing cell therapy;

administering to a non-responder or partial responder, for example, an additional agent in combination with a CAR-expressing cell therapy (e.g., a checkpoint inhibitor, e.g., a checkpoint inhibitor as described herein);

Administering to a non-responder or a partial responder a therapy that increases the number of younger T cells in the subject prior to the CAR-expressing cell therapy treatment;

for example, for a subject identified as a non-responder or partial responder, a process of production of a cell therapy expressing a CAR is adjusted, e.g., younger T cells are enriched prior to introduction of the nucleic acid encoding the CAR, or transduction efficiency is increased;

for example, to non-responders or partially responders or relapsers, alternative therapies are administered; or

If the subject is or is identified as a non-responder or relapser, reducing T_REGCell population and/or T_REGGene signature, e.g., by depletion of one or more CD25, administration of cyclophosphamide, an anti-GITR antibody, or a combination thereof.

In certain embodiments, the subject is pre-treated with an anti-GITR antibody. In a certain embodiment, the subject is treated with an anti-GITR antibody prior to infusion or reinfusion.

Biopolymer delivery method

In some embodiments, one or more CAR-expressing cells as disclosed herein can be administered or delivered to a subject via a biopolymer scaffold, e.g., a biopolymer implant. The biopolymer scaffold can support or enhance the delivery, expansion, and/or dispersion of CAR-expressing cells described herein. The biopolymer scaffold comprises a biocompatible (e.g., does not substantially induce an inflammatory response or immune response) and/or biodegradable polymer that can be naturally occurring or synthetic.

Examples of suitable biopolymers include, but are not limited to, agar, agarose, alginate/Calcium Phosphate Cement (CPC), beta-galactosidase (beta-GAL), (1,2,3,4, 6-pentaacetoacetate-D-galactose), cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite, poly (3-hydroxybutyrate-co-3-hydroxy-hexanoate) (phbhfx), poly (lactide), poly (caprolactone) (PCL), poly (lactide-co-glycolide) (PLG), polyethylene oxide (PEO), poly (lactic-co-glycolic acid) (PLGA), polypropylene oxide (PPO), and mixtures thereof, alone or in any concentration and in any ratio in combination with any other polymer component, Polyvinyl alcohol) (PVA), silk, soy protein, and soy protein isolate. The biopolymer may be enhanced or modified with molecules that promote adhesion or migration, such as collagen mimetic peptides that bind to collagen receptors of lymphocytes and/or stimulating molecules that enhance delivery, expansion, or function (e.g., anti-cancer activity) of the cells to be delivered. The biopolymer scaffold can be an injectable, e.g., a gel or semi-solid, or a solid composition.

In some embodiments, CAR-expressing cells described herein are plated onto a biopolymer scaffold prior to delivery to a subject. In embodiments, the biopolymer scaffold further comprises one or more additional therapeutic agents described herein (e.g., another CAR-expressing cell, an antibody, or a small molecule) or a substance that enhances the activity of the CAR-expressing cells, e.g., a biopolymer incorporated into or conjugated to the scaffold. In embodiments, the biopolymer scaffold is injected (e.g., intratumorally injected) or surgically implanted at or near the tumor sufficient to mediate the anti-tumor effect. Additional examples of biopolymer compositions and methods for their delivery are described in Stephan et al, Nature Biotechnology,2015,33: 97-101; and WO 2014/110591.

Pharmaceutical compositions and treatments

The pharmaceutical compositions of the invention can comprise a CAR-expressing cell as described herein, e.g., a plurality of CAR-expressing cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline, and the like; sugars such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. In one aspect, the compositions of the present invention are formulated for intravenous administration.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the condition of the patient and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

In one embodiment, the pharmaceutical composition is substantially free of (e.g., absent of) detectable levels of impurities, e.g., selected from endotoxin, Mycoplasma (Mycoplasma), Replication Competent Lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD 3/anti-CD 28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cells or plasmid components, bacteria, and fungi. In one embodiment, the bacteria is at least one selected from the group consisting of: alcaligenes faecalis (Alcaligenes faecalis), Candida albicans (Candida albicans), Escherichia coli (Escherichia coli), Haemophilus influenzae (Haemophilus influenza), Neisseria meningitidis (Neisseria meningitidis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Staphylococcus aureus (Staphylococcus aureus), Streptococcus pneumoniae (Streptococcus pneumoniae), and Streptococcus pyogenes (Streptococcus pyogenes) group A.

When an "immunologically effective amount", "anti-tumor effective amount", "tumor inhibiting effective amount", or "therapeutic amount" is indicated, the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and patient (subject) condition. It can be stated generally that a pharmaceutical composition comprising T cells as described herein can be as 10⁴To 10⁹Individual cells/kg body weight, in some cases 10⁵To 10⁶Doses of individual cells/kg body weight (including all integer values within these ranges) are administered. The T cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med.319:1676,1988).

In certain aspects, it may be desirable to administer activated T cells to a subject and then subsequently withdraw blood (or perform apheresis), activate T cells therefrom according to the invention and return these activated and expanded T cells to the patient. This process may be performed many times every few weeks. In certain aspects, T cells from 10cc to 400cc of blood draw may be activated. In certain aspects, T cells from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc blood draws are activated.

Administration of the subject compositions may be carried out in any convenient manner, including aerosol inhalation, injection, ingestion, blood transfusion, implantation, or transplantation. The compositions described herein may be administered to a patient intra-arterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one aspect, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In one aspect, a composition of CAR-expressing cells (e.g., T cells or NK cells) of the invention is administered by intravenous injection. A composition of cells expressing the CAR (e.g., T cells or NK cells) can be injected directly into a tumor, lymph node, or site of infection.

In one particular exemplary aspect, the subject may be subjected to leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate cells of interest, e.g., immune effector cells (e.g., T cells or NK cells). These immune effector cell (e.g., T cell or NK cell) isolates can be expanded and treated by methods known in the art such that one or more CAR constructs of the invention can be introduced, thereby generating a CAR-expressing cell of the invention (e.g., a CAR T cell or a CAR-expressing NK cell). Subjects in need thereof may then receive standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain aspects, following or concurrent with transplantation, the subject receives an infusion of expanded CAR-expressing cells of the invention (e.g., T cells or NK cells). In an additional aspect, the expanded cells are administered before or after surgery.

In embodiments, the subject is subject to lymphocyte depletion, e.g., prior to administration of one or more cells expressing a CAR described herein (e.g., a CAR that binds CD33 described herein). In embodiments, lymphocyte depletion comprises administration of one or more of: melphalan, cyclophosphamide (cytoxan), cyclophosphamide and fludarabine.

The dosage of the above-described therapies to be administered to a patient will vary with the exact nature of the condition being treated and the recipient of the treatment. The magnification of the dosage administered to humans can be performed according to accepted practice in the art. For adult patients, the dose of CAMPATH, for example, will typically range from 1 to about 100mg, typically administered daily for a period of between 1 day and 30 days. A preferred daily dose is 1 to 10mg per day, although in some cases larger doses up to 40mg per day are used (as described in us patent No. 6,120,766).

In one embodiment, the CAR is introduced into an immune effector cell (e.g., a T cell or NK cell), e.g., using in vitro transcription, and the subject (e.g., a human) receives an initial administration of a CAR immune effector cell of the invention (e.g., a T cell or NK cell) and one or more subsequent administrations of a CAR immune effector cell of the invention (e.g., a T cell or NK cell), wherein the one or more subsequent administrations are less than 15 days after the previous administration, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, a CAR immune effector cell (e.g., T cell or NK cell) of the invention is administered to a subject (e.g., human) more than once per week, e.g., a CAR immune effector cell (e.g., T cell or NK cell) of the invention is administered 2, 3, or 4 times per week. In one embodiment, a subject (e.g., a human subject) receives more than one administration (e.g., 2, 3, or 4 administrations per week) (also referred to herein as a cycle) of CAR immune effector cells (e.g., T cells, NK cells) per week, followed by one week without administration of CAR immune effector cells (e.g., T cells, NK cells), and followed by one or more additional administrations (e.g., more than one administration of CAR immune effector cells (e.g., T cells, NK cells) per week) of CAR immune effector cells (e.g., T cells, NK cells) to the subject. In another embodiment, the subject (e.g., human subject) receives more than one cycle of CAR immune effector cells (e.g., T cells, NK cells) and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the CAR immune effector cells (e.g., T cells, NK cells) are administered 3 times weekly every 1 day. In one embodiment, the CAR immune effector cells (e.g., T cells, NK cells) of the invention are administered for at least two, three, four, five, six, seven, eight or more weeks.

In one aspect, a lentiviral vector (e.g., a lentivirus) is used to generate a cell that expresses a CD33CAR (e.g., a CD33 CART or a NK cell that expresses a CD33 CAR). CAR-expressing cells (e.g., CART or CAR-expressing NK cells) generated in this manner will have stable CAR expression.

In one aspect, a viral vector (such as a gamma retroviral vector, e.g., a gamma retroviral vector described herein) is used to generate a CAR-expressing cell, e.g., a CART. CART generated using these vectors can have stable CAR expression.

In one aspect, the CAR-expressing cell (e.g., CART or CAR-expressing NK cell) transiently expresses the

CAR vector

4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. Transient expression of the CAR can be achieved by delivering an RNA CAR vector. In one aspect, the CAR RNA is transduced into a cell (e.g., a T cell or NK cell) by electroporation.

A potential problem that can arise in patients treated with cells that transiently express a CAR (e.g., CART or CAR-expressing NK cells), particularly with CAR-expressing cells that carry a murine scFv (e.g., CART or CAR-expressing NK cells), is allergy following multiple treatments.

Without being bound by this theory, it is believed that this allergic response may be caused by the patient developing an anti-CAR humoral response (i.e., an anti-CAR antibody with anti-IgE isotype). It is believed that in the presence of a 10 to 14 day antigen exposure off-period, antibody-producing patient cells undergo a class switch from the IgG isotype (which does not cause allergy) to the IgE isotype.

The CAR-expressing cell (e.g., CART or CAR-expressing NK cells) infusion break should not last for more than 10 to 14 days if the patient is at high risk of developing an anti-CAR antibody response during the course of transient CAR therapy (such as those produced by RNA transduction).

Examples

The invention is further detailed by reference to the following experimental examples. These embodiments are provided for the purpose of illustration only and are not intended to be limiting unless otherwise specified. Thus, the present invention should not be construed as limited in any way by the following examples, but rather should be construed to cover any and all modifications that become apparent from the teachings provided herein.

Without further description, it is believed that one of ordinary skill in the art, using the preceding description and the following illustrative examples, can make and use the compounds of the present invention and practice the claimed methods. The following working examples specifically point out various aspects of the invention and are not to be construed as limiting the remainder of the disclosure in any way.

Example 1: humanized CAR constructs

CD33 levels were measured by flow cytometry in primary proprietary samples of AML patients using commercially available antibodies (clone HIM3-4, eBioscience; or clone WM53, Biolegend). The results provided herein demonstrate that CD33 is expressed in a number of primary AML patient samples (using standard side scatter) ^{Is low in}CD45^DarknessCharacterized by gating AML blasts); n-35-46 per group).

A schematic of the CAR construct used in this example is shown in figure 3. All are second generation CARs using 41BB and CD3 zeta signaling. The scFv of CART33 was derived from clone MY 9-6.

In vitro Activity of CART33

The experiments described herein measured CART 33-mediated T cell degranulation. CART33 transduced T cells and UTD T cells were incubated with the CD33+ cell line MOLM14 and the control ALL cell line NALM6 and CD107a degranulation was measured by flow cytometry. Expression of both the murine CART33 construct and the humanized CART33 construct elicited specific degranulation in the presence of MOLM14 (P <0.001) (fig. 4).

The experiments described herein measured cytokine production in response to CART 33. Both humanized and murine CART33 expressing T cells produced cytokines upon incubation with MOLM14 (figure 5). Intracellular tumor necrosis factor alpha and interferon gamma were measured by flow cytometry.

The experiments described herein measured the proliferation of CART123 expressing T cells and CART33 expressing T cells. The proliferation of humanized CART33 and murine CART33 in response to MOLM14 was measured. The results are shown in fig. 6. T cells were labeled with CFSE and incubated for 120 hours under control conditions or with MOLM 14. Non-proliferating T cells retained a single bright peak of CFSE expression (based on green fluorescence in the FITC channel), whereas proliferation of CART cells had more than one CFSE peak and expression below baseline.

The experiments described herein measured specific killing of CART123 expressing T cells, humanized CART33 expressing T cells, and murine CART33 expressing T cells. T cells were incubated with MOLM14 or NALM6 (controls) for 24 hours. At low E: T ratios, expression of huCART33 resulted in significantly more specific killing compared to murine CART33 (fig. 7). Killing is measured using flow Cytometry-based assays after CFSE-labeling of tumor cells (e.g., Cao et al, Cytometry Part A2010; 7& A: 534-. In the latter assay, the number of viable target cells is positively correlated with the number of photons emitted.

After 24 h incubation with T cell medium only, PMA/ionomycin, MOLM14 or NALM6 (control cell lines), cytokine profiles of T cells expressing humanized CART33, murine CART33, CART123 were determined using the 30-plex Luminex kit (Invitrogen) (fig. 8).

CART33(IgG4 hinge) and CART33(CD8 hinge) have equivalent in vitro activity

The experiments described herein measure degranulation. CART33(IgG4 hinge), CART33(CD8 hinge), CART123 and non-transduced T cells were incubated with the CD33+ cell line MOLM14 and CD107a degranulation was measured by flow cytometry. The results presented herein show that both CART33 constructs underwent specific degranulation in the presence of MOLM14 (fig. 8).

The experiments described herein measure cytokine production. After incubation with MOLM14 cell line, both the CART33 construct and CART123 specifically induced cytokine production (fig. 9). Intracellular tumor necrosis α, MIP1a, and interferon γ were measured by flow cytometry.

The experiments described herein measured the proliferation of untransduced controls, CART33(IgG4 hinge) -expressing T cells, CART33(CD8 hinge) -expressing T cells, or CART 123-expressing T cells in response to MOLM14 (figure 10). T cells were labeled with CFSE and incubated with MOLM14 for 120 hours. Proliferation was measured by CFSE dilution. Non-proliferating T cells were brightly stained with CFSE and showed a single peak. One cell division is considered as two peaks, two cell divisions are considered as three peaks, etc.

In vivo antitumor Activity of equivalents of CART33-CD8H, CART33-IgG4H, and CART123

To compare the in vivo anti-tumor effects of CART33-CD8H, CART33-IgG4H, and CART123, NOD-SCID-common gamma chain knockout (NSG) mice were injected intravenously with 1x10⁶Individual AML cell lines MOLM14 and imaged for implantation after 6 days. On day 7, mice were treated with T cells expressing CART33(IgG4 hinge), CART33(CD8 hinge), CART123, or control vehicle (untransduced cells). The total number of injected T cells was 2x10 ⁶And IV. Mice were followed weekly for continuous imaging to assess tumor burden (figure 11).

Data on tumor burden over time were obtained by bioluminescence imaging (BLI). Data were from one experiment (5 per group) and a representative of 4 independent mouse experiments is shown in figure 12. The results provided herein demonstrate the equivalent in vivo anti-tumor effects of CART33-CD8H, CART33-IgG4H, and CART123T cells.

CART33 and CART123 produce equivalent primary AML eradication in vivo

To compare the primary AML eradication effects of CART33 and CART123 in vivo, primary AML samples were tested at 5x10⁶Individual cells were injected intravenously into human cytokine IL3/GM-CSF/SCF transgenic NSG mice (NSGS mice). Implantation was confirmed by retro-orbital bleeding after 2-4 weeks and mice were subsequently treated with CART33, CART123, or control vehicle (untransduced cells). The total number of injected T cells was 1x10⁵And i.v. Mice were followed by a series of retro-orbital bleeds to assess AML burden (figure 13).

Peripheral blood analyses from UTD, CART33 or CART123 treated mice were performed at baseline, day 14 and day +70 (fig. 14). Blood was obtained from the retro-orbital sinus of anesthetized mice using standard techniques. Subsequently, a standard volume of 50-60ul of blood was lysed in 1ml of ACK lysis buffer. The blood was then stained with a fluorescently labeled antibody and the presence of AML or CART cells was detected using flow cytometry. AML was not detected in CART33 or CART123 treated mice.

Disease burden was measured at different time points with the aid of blast cells/ul from retroorbital bleeds (fig. 15).

Survival of mice treated with CART33, CART123, or UTD (p <0.001 when CART33 or CART123 was compared to UTD) was measured (figure 16). The results provided herein demonstrate that CART33 and CART123 produce equivalent primary AML eradication in vivo.

Hematopoietic stem cell toxicity of CART33 cells

To determine hematopoietic stem cell toxicity of CART33 cells, blood was collected retro-orbitally from Humanized Immune System (HIS) mice 6-8 weeks after injection of human CD34+ cells derived from fetal liver to confirm engraftment of human cells. The mice were then administered CART33 or UTD (1 x10 each)⁶Individual cells) and weekly continuous retro-orbital blood sampling. Mice were then euthanized on day 28 and organs were harvested and analyzed (fig. 17).

Peripheral blood analysis was performed by flow cytometry (via retro-orbital bleeding) from day 28 on completion of the experiment (fig. 18). Statistical analysis of day 28 peripheral blood analysis from CART33 treated, UTD or untreated (n ═ 5) mice showed that CART33 was specific for monocytes and CD33⁺Myeloid cells produced significant toxicity with B cells and platelets being rare (fig. 19). Bone marrow analysis by flow cytometry at day 28 showed that CART33 treatment resulted in a significant reduction in single, huCD45 dark, lineage-negative gated myeloid progenitor cells (CD34+ CD38+) and hematopoietic stem cells (CD34+ CD38-) (fig. 20).

Sections of the femur were taken from mice at day 28 after UTD T cell or CART33 cell treatment. huCD45 and CD34 staining was performed by IHC (fig. 21). There was no difference in huCD45 between control T cells and CART33, although both groups showed less huCD45 staining, which is likely consistent with an allogeneic human-anti-human effect. There was a specific reduction of CD34+ cells in CART33 treated mice. The results are representative of two experiments.

CART33 and CART123 produce equivalent hematopoietic toxicity in vivo

To determine the in vivo hematopoietic toxicity of CART33 and CART123, NSGS mice received intraperitoneal administration of busulfan followed by the next day 2x10 from normal donors⁶Individual T cell depleted bone marrow cells. Implantation was confirmed by flow cytometric analysis of peripheral blood after 4 weeks, and subsequently with 1x10⁶Mice were treated with autologous T cells treated with CART33, CART123, or UTD. Followed byMice were followed by retroorbital bleeding on

days

7 and 14 and were euthanized for necropsy on day 14 (fig. 22).

Bone marrow analysis was performed by flow cytometry at day 28. CART33 and CART123 treatments resulted in a significant reduction in huCD45 dark, Lin-gated myeloid progenitor cells (CD34+ CD38+) and hematopoietic stem cells (CD34+ CD 38-). Results are representative of two experiments (fig. 23). The results provided herein suggest that CART33 and CART123 produce equivalent hematopoietic toxicity in vivo.

CD 34-enriched BM from MDS patients were incubated with UTD, CART33(IgG4 hinge), CART33(CD8 hinge), or CART 123. CD45 dark CD34+ cells were significantly reduced in CART33 or CART123 treated samples (fig. 24). The results provided herein demonstrate that CART33 and CART123 are cytotoxic myelodysplastic syndrome (MDS) bone marrow cells.

Additional experiments to examine the activity of humanized CART33 are described in examples 5-6.

Example 2: CAR constructs

Fully human anti-CD 33 single-chain variable fragment (scFv) was generated and cloned into a lentiviral expression vector along with intracellular CD3 zeta chain and intracellular costimulatory domain 4-1BB and given the names described in table 1 (which is shown in the detailed description of the invention).

The order in which the VL and VH domains occur in the scFv varies (i.e., VL-VH, or VH-VL orientation), and wherein 3 or 4 copies of a "G4S" (SEQ ID NO:25) subunit (wherein each subunit comprises the sequence GGGGS (SEQ ID NO:25) (e.g., (G4S)₃(SEQ ID NO:28) or (G4S)₄(SEQ ID NO:27)) to generate the integrity of the scFv domains as shown in Table 3.

The sequences of human scFv fragments (SEQ ID NOS: 39-83, including optional leader sequences) are provided in Table 2 herein (in the detailed description of the invention). The sequences of human scFv fragments without leader sequence (nucleotide sequence SEQ ID NO:255-261, and amino acid sequence SEQ ID NO:262-268) are provided herein in Table 9 of the detailed description of the invention.

These clones all contained a Q/K residue change in the signal domain derived from the costimulatory domain of the CD3 zeta chain. The CAR scFv fragment was subsequently cloned into a lentiviral vector to generate a full length CAR construct in a single coding open reading frame and using the EF1 a promoter for expression (SEQ ID NO: 11).

The sequence of the CAR construct and its domain sequences are listed in the detailed description of the invention. Analysis of human CAR constructs was performed as described in example 4.

Example 3: humanized CART sequences

2213 mouse anti-CD 33IgG4 nucleotide sequence (SEQ ID NO:138)

GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGCTGCATACGCGTCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGTGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGCTAGCTCTAGAGCCACCATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCCGGATCCAACATCATGCTGACCCAGAGCCCTAGCAGCCTGGCCGTGTCTGCCGGCGAGAAAGTGACCATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGAGCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCGGAGGCACCAAGCTGGAAATCAAGAGAGGCGGCGGAGGCTCAGGCGGAGGCGGATCTAGTGGCGGAGGATCTCAGGTGCAGCTGCAGCAGCCAGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACGACGACATCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGAGCCGGCACCACCGTGACCGTGTCATCTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATGATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG

2213 CAR nucleotide sequence (SEQ ID NO:139)

ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCCGGATCCAACATCATGCTGACCCAGAGCCCTAGCAGCCTGGCCGTGTCTGCCGGCGAGAAAGTGACCATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGAGCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCGGAGGCACCAAGCTGGAAATCAAGAGAGGCGGCGGAGGCTCAGGCGGAGGCGGATCTAGTGGCGGAGGATCTCAGGTGCAGCTGCAGCAGCCAGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACGACGACATCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGAGCCGGCACCACCGTGACCGTGTCATCTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATGATCTACATC

TGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

2213 CAR amino acid sequence (SEQ ID NO:140)

MALPVTALLLPLALLLHAARPGSNIMLTQSPSSLAVSAGEKVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQSEDLAIYYCHQYLSSRTFGGGTKLEIKRGGGGSGGGGSSGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFKGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGAGTTVTVSSSGESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

2213scFv nucleotide sequence (SEQ ID NO:141)

ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCCGGATCCAACATCATGCTGACCCAGAGCCCTAGCAGCCTGGCCGTGTCTGCCGGCGAGAAAGTGACCATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGAGCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCGGAGGCACCAAGCTGGAAATCAAGAGAGGCGGCGGAGGCTCAGGCGGAGGCGGATCTAGTGGCGGAGGATCTCAGGTGCAGCTGCAGCAGCCAGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACGACGACATCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGAGCCGGCACCACCGTGACCGTGTCATCT

2213scFv amino acid sequence (SEQ ID NO:142)

MALPVTALLLPLALLLHAARPGSNIMLTQSPSSLAVSAGEKVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQSEDLAIYYCHQYLSSRTFGGGTKLEIKRGGGGSGGGGSSGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFKGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGAGTTVTVSS

2218 humanized anti-CD 33IgG4H nucleotide sequence (SEQ ID NO:143)

GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGCTGCATACGCGTCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGTGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGCTAGCTCTAGAGCCACCATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCCGGATCCGAGATCGTGCTGACACAGAGCCCTGGAAGCCTGGCCGTGTCTCCTGGCGAGCGCGTGACAATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAGACTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGCCCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGAGAGGCGGCGGAGGCTCTGGCGGAGGCGGATCTAGTGGCGGAGGATCTCAGGTGCAGCTGCAGCAGCCTGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACGACGACATCAGCTACAACCAGAAGTTCCAGGGCAAGGCCACCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGCCAGGGAACCACCGTGACCGTGTCATCTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATGATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG

Humanized myy 96(L2H) IgG4H BBz NT (SEQ ID NO:144)

ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCCGGATCCGAGATCGTGCTGACACAGAGCCCTGGAAGCCTGGCCGTGTCTCCTGGCGAGCGCGTGACAATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAGACTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGCCCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGAGAGGCGGCGGAGGCTCTGGCGGAGGCGGATCTAGTGGCGGAGGATCTCAGGTGCAGCTGCAGCAGCCTGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACGACGACATCAGCTACAACCAGAAGTTCCAGGGCAAGGCCACCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGCCAGGGAACCACCGTGACCGTGTCATCTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATGATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

Humanized myy 96(L2H) IgG4H BBz AA (SEQ ID NO:145)

MALPVTALLLPLALLLHAARPGSEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGGGGSGGGGSSGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSSGESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Humanized my96(L2H) scFv nt (SEQ ID NO:146)

GAGATCGTGCTGACACAGAGCCCTGGAAGCCTGGCCGTGTCTCCTGGCGAGCGCGTGACAATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAGACTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGCCCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGAGAGGCGGCGGAGGCTCTGGCGGAGGCGGATCTAGTGGCGGAGGATCTCAGGTGCAGCTGCAGCAGCCTGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACGACGACATCAGCTACAACCAGAAGTTCCAGGGCAAGGCCACCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGCCAGGGAACCACCGTGACCGTGTCATCT

Humanized my96(L2H) scFv AA (SEQ ID NO:147)

EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGGGGSGGGGSSGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSS

Example 4: human CAR constructs

To describe cell surface expression of scFv on Jurkat T cell line containing a luciferase reporter driven by NFAT regulated promoter (referred to as JNL cells), JNL cells were transduced with lentiviral vectors expressing cDNA encoding GFP, scFv binding to CD19 or cDNA encoding scFv generated against CD33 (figure 27). Cell surface expression of individual scfvs on JNL was detected by incubating cells with Fc-tagged recombinant hsCD33, followed by an Fc-specific secondary antibody conjugated to phycoerythrin. Clones CD33-1, -2, -3, -4, -5, -6, -7, and-9 were observed to bind to hsCD33 at appreciable levels relative to JNL cells expressing GFP or scFv targeting CD19, which served as negative controls for this assay. CD33-8 was also observed to lack evaluable binding to hsCD33 or protein L (bacterial cell surface protein that binds immunoglobulin light chains) (data not shown). The data presented herein show that clones CD33-1, -2, -3, -4, -5, -6, -7, and-9 encode scFv that bind to hsCD 33.

Individual scfvs targeting hsCD33 were evaluated for their ability to stimulate NFAT activity in JNL cells (figure 28). JNL cells expressing scFv to hsCD33 were co-cultured with MOLM13 or MOLP8 cell lines expressing hsCD33 or lacking hsCD33 cell surface expression, respectively (FIG. 28A; hsCD33, green solid line; isotype control, grey dashed and shaded area). Figure 28B depicts activation of JNL cells expressing scFv targeting hsCD33 in the presence of MOLM13 cells (solid line) or MOLP8 cells (dashed line). JNL cells expressing the respective scFv were plated at different effector (i.e., JNL cells) to target (i.e., MOLM13 or MOLP8) ratios and Bright-Glo was used on an EnVision instrument 24 hours after incubation^TMLuciferase assays analyze expression relative to luciferase units (RLU). It was observed that scFv clones CD33-1, -2, -3, -4, -5, -6, -7, -9 and scFv based on the sequence of monoclonal antibody Mylotarg binding to human CD33 antigen (CD33-UPenn) were able to trigger NFAT-dependent luciferase activity in the presence of MOLM13, although to varying degrees, relative to MOLP8 or GFP-expressing JNL cells.

The activity of scFv targeting hsCD33 was further assessed in donor-derived primary T cells (fig. 29A and 29B). Naive T cells were isolated from healthy donor PBMCs by negative selection using standard protocols and Use of

Human T-Expander CD3/CD28 Invitrogen kit activation. After 24 hours post-stimulation, T cells were transduced with lentiviruses expressing individual scfvs targeting hsCD33 and expanded in culture for an additional 9 days. T cell cultures were analyzed for their ability to undergo cell expansion and to stimulate cytolytic activity in an antigen-dependent and antigen-independent manner. Expression of scFv on the cell surface of lentiviral vector-transduced human primary T cells is depicted in fig. 29A and 29B, which express scFv targeted to hsCD 33. Expression of scFv was detected by incubating the cells with Fc-tagged recombinant hsCD33 and an Fc-specific secondary antibody conjugated to phycoerythrin as described in figure 27. Consistent with the foregoing results in FIG. 27, scFv clones CD33-1, -2, -3, -4, -5, -6, -7, and-9 were expressed at evaluable levels in primary T cells that bound to hsCD33 relative to T cells expressing GFP that lacked the ability to bind to hsCD 33. Thus, scFv clones CD33-1, -2, -3, -4, -5, -6, -7, and-9 can be used to engineer primary T cells to target the human CD33 antigen.

T cells were labeled with CFSE and co-cultured or cultured alone (fig. 30A, shaded bars) in the presence of MOLM13 (fig. 30A, solid black line), MOLP8 (fig. 30A, open white bars) to assess the proliferative capacity of UTD primary T cells or cells expressing scFv targeting hsCD 33. It was observed that T cells expressing scFv clones CD33-2, -3, -4, -5, -6 and-9 were able to expand in an antigen-dependent manner, as evidenced by the increased absolute number of T cells in the presence of MOLM13 cells expressing CD33 compared to UTD or CD33 negative MOLP8 cells (fig. 30A). Furthermore, antigen-driven cell division in primary T cells was assessed by measuring the Median Fluorescence Intensity (MFI) of CFSE-labeled T cells co-cultured with MOLM13 cells, MOLP8 cells, or alone, which expressed scFv clones CD33-2, -3, -4, -5, -6, and-9 (fig. 30B). Consistent with the aforementioned increase in T cell expansion (fig. 30A), a reduction in CFSE levels was observed in T cells co-cultured with MOLM13 compared to UTD cells or MOLP8 cells (fig. 30B). For scFv ⁺Or scFv^-When the T cell is gated, hair is sentNow co-cultured with MOLM13, scFv expressing T cells versus their scFv^-The counterpart had an attenuated CFSE level. However, scFv co-cultured on MOLP8 cells⁺T cells or scFv^-CFSE levels were similar between T cells (fig. 30B), indicating increased proliferation in response to antigen recognition. The results presented herein are also consistent with the ability of scFv clones CD33-2, -3, -4, -5, -6, and-9 to stimulate scFv-dependent JNL cell activation in the presence of CD33 (fig. 28B). Thus, scFv clones CD33-2, -3, -4, -5, -6, and-9 can be used to activate proliferation of primary T cells in a CD 33-dependent manner.

In addition, CART-CD33T cells were tested for their ability to proliferate in response to exposure to antigens on target cells. Target cells include PL-21, HL60 and Molp8 cells. On the day of analysis (day 0), target cells were counted and transferred to 6mL T cell culture medium in 50mL tubes at 3e6 cells/mL. Target cells were irradiated at 10,000rad on ice. After irradiation, the target cells were washed 2 times in T cell culture medium, counted and resuspended to 5e5 cells/ml in T cell culture medium on ice. Frozen transduced T cells were thawed, washed in 10mL T cell complete medium, centrifuged at 300g for 10min and gently resuspended in 3mL T cell complete medium at room temperature. The T cells were then counted in a cell counter and resuspended to 2.5e6/mL in 10mL of media. In 96-well U-bottom plates, 25,000 irradiated target cells and 25,000 transduced CAR T cells (1:1 ratio) were pooled in duplicate wells. In separate wells, add 75,000 anti-CD 3/CD28 beads to 25,000 transduced T cells in 100 μ Ι of medium to generate a 1:3 cell to bead ratio as a positive control; in another well, only 100 μ Ι of medium to 25,000 transduced T cells were added as a control containing medium only. Cells were incubated at 37 ℃ with 5% CO ₂Incubate for 4 days. On day 4, cells were harvested and duplicate cultures were pooled by aspiration and transferred to the same well on a U-shaped bottom plate to stain FACS for CD4, CD8 and CAR using protein L or recombinant human CD33 protein. After staining, cells were resuspended in 120 μ l MACS + 0.5% BSA buffer and 20 μ l/well Countbright beads were added to each well. Proliferation is determined as detected over a period of time used to count 2500 beadsFACS positive cell number (fig. 47). The tested CART-CD33T cells (e.g., derived from scFv clone CD33-1, -2, -4, -5, -6, -7, -9, and humanized My96 (referred to as "Upenn") proliferated to a greater extent when exposed to HL-60 target cells than either the untransduced cells or CART-CD19T cells when exposed to PL21 target cells, CART-CD33T cells (e.g., derived from scFv clone CD33-1, -2, -4, -5, -6, -7, -9 and humanized My96 (referred to as "Upenn") proliferated to a greater extent than non-transduced cells and proliferated to a degree approximately equal to or greater than CART-CD19T cells when exposed to MOLP8 cells that do not express the CD33 target, CART-CD33T cells proliferated to a degree approximately the same as or slightly greater than non-transduced cells or CART-CD19T cells.

To assess cytolytic activity, 25,000 MOLM13 cells were plated at different effector (i.e., T cell) to target (i.e., MOLM13) ratios with primary T cells expressing each scFv and the extent of killing by MOLM13 was analyzed by counting the absolute number of CFSE labeled MOLM13 cells after 4 days of culture (fig. 31). It was observed that scFv clones CD33-2, -3, -4, -5, -6 and-9 were able to induce lysis of target cells, although to varying degrees. Thus, scFv clones CD33-2, -3, -4, -5, -6, and-9 can be used to engineer primary T cells to directly target and kill target cells expressing CD 33.

T cell killing was directed to PL21 expressing CD33 and HL-60 acute myeloid leukemia cell line stably expressing luciferase. Non-specific background killing levels were determined using U87 cells that do not express CD33 as a control and using untransduced T cells (UTD). Cytolytic activity of CART-CD33 was measured as a titration of the effector to target cell ratio of 10:1 and 2-fold decreasing dilution of T cells, where the effector is defined as T cells expressing the anti-CD 33 chimeric receptor. The assay is initiated by mixing an appropriate number of T cells with a constant number of target cells. After 20 hours, Bright-Glo was used^TMLuciferase assay luciferase signal was measured on an EnVision instrument. Comparing these killing curves, titrating the amount of effector cells showed that those expressing CD33 were destroyed (fig. 48A, 48B, and 48C). T cells from the same donor transduced with any CAR-CD33 cell carrying human scFv were able to selectively kill CD33+ target, although The difference in activity is significant, which may translate into a difference in clinical activity.

CART-CD33 cells were tested for their ability to produce cytokines in response to antigens. Cells were thawed and allowed to recover overnight. Untransduced T cells (UTD) were used as a non-specific control for background T cell effects. These T cells were directed against HL-60, PL21 or MOLP8 cells. This assay tests an effector to target ratio of 2.5:1, where an effector is defined as a T cell expressing an anti-CD 33 CAR. After mixing the cells, the assay was run for 16 hours, at which time the media was removed to analyze the cytokines IFN-. gamma., TNF-. alpha.and IL-2 using the CBA-Flex kit for detection of human cytokines. When CART-CD33T cells were cultured with cancer cells endogenously expressing CD33, all CD33-CART produced cytokines in response to cells expressing the target (fig. 49). The differences between the responsiveness of various CD33-CART clones to target cells that express CD33 can translate into better clinical efficacy of CART cells transduced with these constructs.

Finally, scFv cross-reactivity to cynomolgus monkey CD33(cyCD33) was assessed by flow cytometry as shown in figure 32. JNL cells transduced with lentiviral vectors expressing scFv to hsCD33 were incubated with Fc-tagged recombinant hsCD33 (red line) or cyCD33 (blue line) followed by an Fc-specific secondary antibody conjugated to phycoerythrin. Although scFv clone CD33-1, -2, -3, -4, -5, -6, -7, -9 and CD33-UPenn were able to bind hsCD33, we noted that only scFv clone CD33-5 appeared to cross-react with both hsCD33 and cyCD33 (FIG. 32).

Example 5: characterization of RNA electroporated CART33 cells

Comparison of CAR33 expression in lentivirus-transduced or RNA-electroporated T cells

The mRNA anti-CD 33 CAR was generated by in vitro transcription, the anti-CD 33 CAR comprising a humanized anti-CD 33scFv and an IgG4 hinge, e.g., SEQ ID NO: 145. T cells were isolated from normal donors and electroporated with CAR33 mRNA. After electroporation, cells were stimulated with CD3/CD28 beads and expanded in culture. CAR33 expression was measured and quantified by flow cytometry at 8 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days post-electroporation (fig. 33A). The results from this experiment showed that RNA electroporation of CAR33 resulted in expression of CAR33 in donor T cells for at least 7 days. Expression was highest (e.g., greater than 60%) up to day 4 after electroporation (fig. 33B). CAR33 expression decreased from day 5-7 post-electroporation.

CAR33 expression from RNA electroporation was compared to CAR33 stable expression from lentiviral transduction. Donor T cells were transduced with a lentiviral construct comprising CAR33 (such as the vector of figure 26) using standard methods. CAR33 expression was measured by flow cytometry and quantified by Mean Fluorescence Intensity (MFI). CAR33 expression was evaluated at 8 hours, 1 day, 2 days, 3 days, and 4 days post-electroporation or transduction for lentivirus transduced CART33 cells (fig. 34A) and for RNA transfected CART33 cells (fig. 34B). The gray peak to the left of the histogram of figure 34 indicates the lack of CAR33 expression, while the increased CAR33 expression grows to the right of the graph. Lentiviral transduction resulted in stable expression of CAR33 in T cells, with CAR33 expression levels remaining unchanged from 8 hours to 4 days post transduction. In contrast, RNA electroporation caused transient expression of CAR33 in T cells. Specifically, CAR33 expression was highest 8 hours to 1 day post-electroporation, and CAR33 expression decreased from 2 days to 4 days post-electroporation.

In vitro cytotoxic Activity

To assess the specific killing activity of RNA electroporated and lentivirus transduced CART33 cells, CART33 cells were incubated for 24 hours with target cells expressing CD33 (e.g., acute myeloid leukemia cell line MOLM14) or CD 33-negative control cells (e.g., mantle cell lymphoma cell line JEKO) at different effector (CART33 cells) to target (CD33 positive or negative cells) ratios. Effector to target ratios were 0:1, 0.125:1, 0.25:1, 0.5:1, 1:1, and 2: 1. The experiments were repeated at

different time points

1, 2, 3 and 4 days after T cell electroporation/transduction. 1 day after electroporation, RNA electroporated CART33 cells showed specific killing starting with an E: T ratio of 0.125:1 (FIG. 35A). Specific killing of CAR33 positive MOLM14 cells was observed 2 days (fig. 35B), 3 days (fig. 35C) and 4 days (fig. 35D) after electroporation of CART33 cells, whereas the E: T ratio of specific killing increased over time, showing a transient nature of specific killing. Lentivirus-transduced CART33 cells showed potent specific killing activity from 1 to 4 days even at lower E: T ratios.

When the results show differently, comparing the specific activity of one particular E: T ratio between day 1 to day 4 after electroporation, the data show that the specific killing effect of RNA electroporated CART33 cells decreases over time after electroporation when the E: T ratio is at 2:1 (figure 36A) or 1:1 (figure 36B). These results demonstrate the transient nature of the specific killing effect of RNA electroporated CART33 cells. In contrast, lentiviral transduced CAR T cells showed stable levels of specific killing from 1-4 days post transduction.

Example 6: humanized CART33 shows potent preclinical activity on human AML and MDS

A second generation CAR was constructed from anti-CD 33scFv (described in example 3; and SEQ ID NO:145) of gemtuzumab ozogamicin (an immunoconjugate targeting CD 33) with 4-1BB and CD3 zeta signaling domains. Here, the preclinical activity of the second generation CAR33 was described and compared to a previously developed CAR targeting CD123 (CAR 123). The results show that CART33 is able to eradicate human acute myeloid lymphoma and myelodysplastic syndrome CD34+ cells while causing significant myelotoxicity in mouse xenografts. Thus, transiently expressed mRNA modified CART33 was also generated to be used in future studies and clinical trials.

The following materials and methods were used in this example:

generation of CART cells

pTRPE anti-CD 33-41BB-CD3 ζ (CAR33) plasmid DNA was generated by cloning a humanized anti-human CD33scFv derived from gemtuzumab ozogamicin in the light chain to heavy chain direction (clone my96) into the murine CART19 plasmid vector described previously.²⁵Normal donor T cells were positively selected from leukocyte isolates (leukapheresis packages) using anti-CD 4 and anti-CD 8 microbeads (Miltenyi), mixed together at a 1:1 ratio and expanded in vitro using anti-CD 3/CD28Dynabeads (Invitrogen, added on day 1 of culture) along with low doses of IL-2. T cells were transduced at multiplicity of infection (MOI)3 on the day following stimulation with lentiviral supernatant from pTRPEmy96-CD33-41BB-CD3 ζ plasmid transfer Stained 293T cells. anti-CD 3/CD28Dynabeads were removed on day 6 and T cells were expanded in T cell culture medium (X-vivo 15 medium supplemented with 5% human serum, penicillin, streptomycin, and Glutamax) for up to 15 days and then frozen for future experiments. Prior to all experiments, T cells were thawed at 37 ℃ and left overnight. The production of CART123 cells was previously described (Gill et al, 2014, Blood,123: 2343-54).

Cells

MOLM14 cell line was obtained from ATCC and maintained in R10 medium (RPMI medium supplemented with 10% fetal bovine serum, penicillin and streptomycin). MOLM 14-luciferase-GFP cells were used in some experiments. Bone marrow samples of de-identified primary human AML and MDS were obtained from the university of pennsylvania stem cells and xenograft core institution. For all functional studies, AML cells were thawed at least 12 hours prior to analysis and left to stand overnight at 37 ℃. MDS bone marrow samples were enriched for CD34+ cells by positive selection using a macSQurant column (Miltenyi).

Flow cytometry analysis

Anti-human antibodies were purchased from Biolegend, eBioscience, or Becton Dickinson. Cells were isolated from in vitro cultures or from animals, washed 1 time in PBS supplemented with 2% fetal bovine serum and stained on ice after blocking Fc receptors. For cell number quantification, Countbright beads (Invitrogen) were used according to the manufacturer's instructions. In all analyses, populations of interest were gated based on forward and side scatter characteristics, followed by single-peak gating, and Live cells were gated using Live Dead Aqua (Invitrogen). Surface expression of anti-CD 33 CAR was detected by staining with Alexa Fluor647 conjugated goat anti-mouse F (ab') 2 antibody from Jackson Immunoresearch. Flow cytometry was performed on a four-color laser Fortessa analyzer (Becton-Dickinson).

T cell functional assay

T cell degranulation assay

Degranulation assays were performed as previously described.²⁶T cells were incubated with target cells at a 1:5 ratio. CD107a, CD28, CD49d and monensin were added at the time of incubation. At 4 hoursThereafter, cells were harvested and stained for CAR expression, CD3, and Live Dead. Cells were fixed and permeabilized, and then stained with intracellular cytokines.

2. Proliferation assay：

T cells were washed and washed at 1x10⁷The/ml was resuspended in 100ul PBS and stained with 100ul CFSE 2.5. mu.M (Life technologies) for 5 minutes at 37 ℃. The reaction was then quenched with cold R10 and the cells were washed 3 times. The target was irradiated at a dose of 100 Gy. T cells were incubated with irradiated target cells at a 1:1 ratio for 120 hours. Cells were then harvested, stained for CD3, CAR, and Live Dead aqua, and Countbright beads (Invitrogen) were added, followed by flow cytometry analysis.

3. Cytotoxic assay

Primary AML samples labelled with luciferase + MOLM14 cells or CFSE were used for cytotoxicity assays as described previously (Cao et al, 2010, Cytometry a,77: 534-545). Briefly, target and effector T cells were incubated at the indicated ratios for 4 hours or 16 hours. The killing effect was calculated by bioluminescence imaging on a Xenogen IVIS-200 spectral camera or by flow cytometry. For the latter, cells were harvested and Countbright beads and 7-AAD were added prior to analysis. The remaining viable target cells were CFSE + 7-AAD-. For MDS, as shown, T cells were incubated with CD 34-enriched bone marrow at a 1:1 ratio for 4 hours or 24 hours, and subsequently cytotoxicity was measured by flow cytometry or by fluorescence in situ hybridization (using probes for specific chromosomal malformations in MDS samples).

4. Cytokine measurement：

As shown, effector cells and target cells were incubated in T cell media at a 1:1 ratio for 24 hours or 72 hours. Supernatants were harvested and analyzed by 30-plex Luminex array according to the manufacturer's protocol (Invitrogen).

In vivo experiments

NOD-SCID-gamma chain-/- (NSG) mice and human IL-3, stem cell factor and GM-CSF (NSG-S) transgenic NSG mice were originally obtained from Jackson Labs. All experiments were performed according to protocols approved by the institutional animal care and use committee of the university of pennsylvania. The scheme of the xenograft model utilized is discussed in detail in the relevant figures and results section. Cells (MOLM14, primary cells or T cells) were injected into the tail vein of mice in 200 μ l PBS at the indicated concentrations. Bioluminescence imaging was performed using a Xenogen IVIS-200 spectral camera. Humanized Immune System (HIS) mice were generated by injecting fetal liver CD34+ cells into neonatal NSG mice, and these mice were used at approximately 8 weeks of age.

Generation of mRNA modified CART33

The CAR construct from pTRPE anti-CD 33-41BB-CD3z plasmid was subcloned into the pDA vector 28 as previously disclosed. Using mMESSAGE

T7 ULTRA transcription kit (Ambion), for in vitro transcription. RNA was purified using RNeasy mini kit (Qiagen). RNA-CAR33 was electroporated into T cells as previously described. Electroporation was performed using ECM830Electro Square Wave Porator (Harvard Apparatus BTX).

Histology and immunohistochemistry

Formalin-fixed, paraffin-embedded sections from mouse femurs were stained with hematoxylin and eosin, counterstained with mabs against human CD45 and human CD34 and captured with a Nikon microscope.

As a result:

CD33 and CART33 constructs as targets in AML

To validate the clinical relevance of CD33 as a target for immunotherapy in AML, the expression level of CD33 in AML was first assessed and found to be expressed on most AML blasts in nearly all primary AML samples (figure 1) as well as in bone marrow from MDS patients (figure 2A, figure 2B, and figure 2C). To assess potential off-target toxicity of CART33, tissue immunohistochemistry was performed on normal tissue stained with 30 parts of anti-CD 33 antibody (LSBio). CD33 was expressed on myeloid lineages of bone marrow and resident macrophages of liver, lung and kidney (fig. 21). To test the efficacy of CAR33, four constructs derived from murine and humanized scFv derived from Gemtuzumab ozogamicin (Gemtuzumab ozogamicin) clone my96 were designed. Two constructs used an IgG4 hinge and two constructs used a CD8 hinge (fig. 3).

CART33 shows potent in vitro activity on AML cell lines, primary AML samples and MDS

The activity of four different CART33 constructs was tested in vitro and compared to CART123 (Gill et al 2014, Blood 123: 2343-. Humanized CART33 consistently outperformed murine CART33 (see example 1) and therefore all subsequent studies were performed on both humanized CAR33 constructs. MOLM14 cell line was used as a model tumor (MOLM14 expresses CD33 and CD 123). Incubation of CART33 with MOLM14 resulted in significant degranulation (fig. 37A), potent cytotoxicity at low effector: target ratios (fig. 37B), extensive proliferation (fig. 37C and 37D), and significantly higher robust cytokine production compared to control T-cell leukemia cell line Jurkat incubation (fig. 38A, fig. 38B, fig. 38C, and fig. 39). After incubation with MOLM14, most of CART33 produced two or more cytokines/cells in a pattern similar to the potent non-specific stimulation of PMA/ionomycin (fig. 38A and 38B). This function has been associated with superior in vivo activity (Carpentio et al, 2009, Proc Natl Acad Sci USA,106: 3360-. In addition, CART33 cells produced more cytokines than CART123 cells (fig. 38C) and also produced significant in vitro activity against primary AML samples. CART33 with an IgG4 hinge gave superior cytotoxicity compared to CART33 with a CD8 hinge (fig. 8, 9, 10, and 40).

CART33 was also tested for in vitro activity in MDS. Bone marrow samples from MDS patients were enriched for CD34 (approximately 85% purity) and subsequently incubated with CART33 cells, CART123 cells, or untransduced control T cells (UTD) at an E: T ratio of 1:5 in the presence of CD49d, CD28 co-stimulation, and monensin for 4 hours. CD107a degranulation was measured by flow cytometry and the percent degranulation was quantified. Fig. 41B and 41C show the specific killing effect of MDS clones with 5q deletion. CD34 enriched bone marrow samples from patients with MDS and 5q loss were incubated with CART33, UTD cells at a 1:1E: T ratio or without treatment for 4 hours. Samples were then harvested and subjected to FISH against 5q- (FIG. 41B). CART33 showed significant in vitro activity in MDS. This is evident by the specific degranulation of CART33 in response to the development of CD 34-enriched MDS samples (fig. 41A), specific killing of CART33 after 24 hours incubation with CD 34-enriched MDS samples (1:1 effector: target ratio, fig. 24), and the specific reduction of malignant clones after 4 hours incubation (measured by FISH) (fig. 41B and fig. 41C).

The results from the in vitro assays described above are summarized in the table below.

TABLE 5 in vitro Activity of CART33 compared to CART123 and untransduced control T cells

All p values when compared to UTD were <0.05

CART33 resulted in a reduction in leukemic burden and survival advantage in MOLM14 implanted xenografts

To test the in vivo activity of CART33, NSG mice were injected with luciferase + MOLM14 (fig. 11). After confirmation of implantation by bioluminescence imaging, mice received a single injection of CART33 or an untransduced T cell control (UTD) at different dose levels. Mice were then followed by continuous imaging and disease burden quantified using bioluminescence. Mice treated with control T cells succumbed to disease rapidly, while mice treated with CART33 showed significant reduction of disease and survival advantage (fig. 42A, 42B, and 42C). In addition, dose response was measured in CART33 treated mice. As shown in the schematic diagram of FIG. 43A, NOD-SCID-gamma chain knockout (NSG) mice were injected intravenously with 1x10⁶Individual AML cell lines MOLM14 and imaged for implantation after 6 days. Mice were treated with CART 335 x10⁶、CART33 2x10⁶、CART33 1x10⁶Or untransduced T cell control 5x10⁶And (6) processing. Mice were followed weekly by continuous imaging to assess AML load. Dose-dependent responses were observed in CART 33-treated mice (fig. 43A and 43B), and superior anti-leukemia activity was observed for CART33 with an IgG4 hinge compared to CAR33 with a CD8 hinge (fig. 44). For all subsequent experiments, only CART33 with an IgG4 hinge was used.

CART33 results in eradication of leukemia in primary AML xenografts and results in long-term disease-free survival

Primary leukemia cells may be more clonal heterogeneous than long-term proliferating cell lines and more fully represent human disease. Thus, efficacy of CART33 in primary AML xenografts was evaluated. NSG-S mice were injected with primary AML samples expressing CD33 and CD 123. Disease burden was quantified in peripheral blood and survival was analyzed (figure 13). Implantation is defined as>1% circulating huCD45+ cells and was generally achieved 2-4 weeks after leukemia cell injection. These mice were then injected with a single injection of CART33 cells, (1 x10 by tail vein injection)⁵Individual) CART123 cells or UTD cells. Leukemia was eradicated within 4 weeks of injection of CART33 or CART123 (fig. 14 and 15) and demonstrated long-term disease-free survival (fig. 16).

CART33 causes the expected cytotoxicity of hematopoietic stem cells and myeloid progenitors

Since CD33 is known to be expressed on myeloid progenitor cells, although at lower levels compared to leukemic cells, the effect of CART33 on normal hematopoiesis was studied. Two different models were used to assess the hematopoietic toxicity of CART 33. Humanized Immune System (HIS) mice post-natally implanted with human fetal CD34+ cells were bled to confirm implantation and subsequently treated with CART33 cells, CART123 cells or untransduced T cells (fig. 17). Mice were bled weekly for 4 weeks. Mice were then euthanized and bone marrow and spleen were harvested from these mice for analysis. As expected based on CD33 expression on myeloid lines, these mice exhibited a reduction in peripheral blood myeloid cells (including monocytes) compared to non-transduced T cell treated mice (fig. 22). Analysis of bone marrow 4 weeks after treatment by flow cytometry (figure 20) or immunohistochemistry (figure 21) showed a reduction in CD34+ CD 38-hematopoietic stem cells and CD34+ CD38+ myeloid progenitor cells. The HIS model is biased towards B-cell lines and thus a second model with a more myeloid bias is used. Here, bone marrow from normal adult donors was depleted of T cells and injected into busulfan-primed NSG-S mice. Autologous CART33, CART123, or UTD was generated by transduction of peripheral blood T cells from bone marrow donors with related lentiviruses. After confirming implantation of these mice, they were treated with autologous CART33, CART123, or UTD and then blood was collected periorbital for a total of 4 weeks (fig. 22). Mice were then euthanized and tissues harvested and analyzed. Similar to HIS xenografts, we observed a reduction in peripheral myeloid and monocytic cells as well as CD34+ bone marrow compartment.

Transient RNA-modified "biodegradable" CART33 results in potent but transient anti-leukemic activity

Since CD33 is expressed on normal hematopoietic cells and tissue resident macrophages (fig. 1 and 2), it is important to validate the safety mechanism prior to clinical use. Thus, RNA-modified CART33 was developed. Electroporation of T cells with RNA-modified CAR-33 resulted in high levels of CAR expression, which was gradually attenuated over 7 days (fig. 33A, fig. 33B, and fig. 34, and as described in example 9). RNA-modified CART33 has similar, but transient, in vitro activity when compared to lentivirus-transduced CART33(LV-CART 33). Incubation of MOLM14 with RNA-CART33 produced specific cytotoxicity comparable to LV-CART33 at E: T ratios 1:1 and 2:1, and which decreased with time after electroporation (fig. 35, and as described in example 9).

RNA-CART33 was tested in combination with in vivo chemotherapy. NSG mice implanted with MOLM14 were treated with cyclophosphamide (60mg/kg I.P) in three doses plus RNA-CART33 combinations or cyclophosphamide plus 10x10⁶Combined treatment of individual UTD cells, given intravenously at three doses (fig. 45). Specifically, NSG mice were injected with MOLM14-luc (1X 10)⁶One intravenous injection) and imaged four days later to confirm implantation. Mice were then randomized to receive either RNA-CART33 in combination with cyclophosphamide or untransduced T cells in combination with cyclophosphamide (60mg/kg I.P). If 10x10 ⁶T cells were administered intravenously at three different doses on

days

5, 9, and 16, and one dose. Cyclophosphamide was given on

days

8 and 14. The combination of cyclophosphamide and RNA modified CART33 resulted in improved leukemia control compared to control mice.

Discussion of the related Art

In this example, the preclinical activity and safety of CART33 in AML was tested, and transiently expressed CART33 was developed and validated as a means to avoid toxicity when used in refractory AML patients. This is the first full preclinical report of CART33 activity, which includes comprehensive survival and toxicity data incorporated into multiple mouse models as well as the anti-leukemic activity of RNA-modified CART 33. CART33 showed potent effector functions on AML cell lines and primary AML samples, including specific killing at low E: T ratios, degranulation, profound proliferation and robust cytokine production, and was also effective in reducing MDS clones after only 4 hours of incubation at 1:1 ratio.

Treatment with CART33 also resulted in eradication of AML and survival advantage in MOLM14 xenografts and primary AML xenografts after a single infusion. The expected myeloid hematopoietic toxicity with CART33 was observed in two different humanized mouse models. Because of the potential bone marrow toxicity and concerns that CD33 is expressed on resident tissue macrophages, RNA-modified CART33 was developed and showed potent but transient in vitro activity. Anti-leukemic effects were also shown by combining RNA-CART33 with chemotherapy.

The experiments described herein and in example 1 also show that for CART33, the use of IgG4 hinges is superior to the use of CD8 hinges. The IgG4 molecule differs significantly from the CD8 molecule. It contains 3 times more amino acids, which may lead to a more flexible hinge region.

Based on the expression of CD33 on leukemic progenitors as well as myeloid progenitors, the hematopoietic toxicity and myeloid progenitor cell depletion and peripheral cytopenia observed in these preclinical studies with CART33 were predicted.

The potential for off-target toxicity with the use of CART33 requires the incorporation of anti-CD 33 therapy that is expressed transiently rather than permanently in clinical trials. RNA-modified CAR have been used in preclinical models (Barrett et al, 2013, Hum Gene Ther,24: 717-727; and Barrett et al, 2011, Hum Gene Ther,22:1575-1586), and phase I experiments with RNA-modified anti-mesothelin CAR T cells in solid tumor patients have shown that this approach is safe and feasible (Beatty et al, 2014, Cancer Immunol Res,2: 112-120). Thus, RNA-modified CART33 was developed as a way to transiently express "biodegradable" CARs to mitigate the possible off-target toxicity of CART 33.

Since a more recent clinical trial showed survival advantage when GO was combined with chemotherapy in low and moderate risk diseases (Hill et al 2014, Lancet oncol.,15: 986-. This combination leads to deeper and longer responses and to a survival advantage for these mice.

These observations highlight several potential translational applications for RNA-modified CART 33. This can be used alone or in combination with chemotherapy in relapsed refractory AML patients who cannot receive allogeneic stem cell transplantation to qualify for transplantation. In addition, RNA-modified CART33 can be incorporated into a conditioning protocol prior to allogeneic stem cell transplantation. Once the safety and feasibility of this approach has been demonstrated in patients, future strategies may include lentiviral CART33 with a "closed" switch. In addition, these findings that both CART33 and CART123 are effective against AML open up a new therapeutic field in combinatorial targeted cell therapy.

Example 7: chimeric antigen receptor T cell therapy against myeloid-derived suppressor cells (MDSCs) in cancer

Recent data show that the myelodysplastic syndrome (MDS) bone marrow microenvironment contains a population of myeloid-derived suppressor cells (MDSCs) expressing CD33 that play an important role in the pathogenesis of MDS through secretion of cytokines that promote ineffective hematopoiesis as well as immunosuppression (Chen et al, J.Clin.Investig.23(2013): 21-223). Chen et al described that MDSC plays a role in inducing myelodysplasia through CD33-S100A9 interaction. This example describes experiments to determine whether MDSCs can be targeted using anti-CD 33 CAR T cell therapy. The results described herein show that aberrant MDS clones and their supporting MDSC populations can be targeted simultaneously with a single anti-CD 33 CAR T cell product.

MDSCs were phenotyped in 4 MDS specimens and 3 normal bone marrow specimens (fig. 50A, fig. 50B, and fig. 50C). For phenotyping, flow cytometry was performed using the following gating strategy: MDSCs were defined as lineage negative (LIN-), HLA-DR negative, CD33 positive cells in MDS bone marrow samples (fig. 50A). MDSC was more abundant in dysplastic bone marrow (MDS-BM) than normal bone marrow (ND-BM) (FIG. 50B). Furthermore, the Mean Fluorescence Intensity (MFI) of CD33 in MDSC population was comparable to CD33 MFI in malignant MDS population and was significantly higher than CD33 MFI in huCD45+ LIN-population from ND-BM (fig. 50C). Thus, CD33 expression in MDSCs was comparable to CD33 expression in the malignant MDS population and was significantly higher than CD33 expression in normal bone marrow. The ability of CART33 to mount responses against MDS blasts and against MDSCs from MDS bone marrow was assessed by quantifying the extent of CD107a degranulation and cytokine production from CART33 cells. CART33 was incubated with negative controls (Jurkat cells), positive controls (PMA and ionomycin), sorted MDS CD34+, or sorted MDSCs from MDS bone marrow for 4 hours. Using the CAR33-UPenn construct (anti-CD 33scFv derived from gemtuzumab ozogamicin), MDS CD34 cells and MDSCs were found to induce broadly equivalent responses in CART33 (fig. 51A and 51B). These results indicate that CART-33, which has a lower affinity, can have differential activity on MDSCs.

MDSCs also play a role in the resistance to immune attack in a variety of other malignancies, including Chronic Lymphocytic Leukemia (CLL), where MDSCs are referred to as nurse-like cells, as well as solid malignancies such as ovarian, breast and colon cancers (Di Mitri et al, Nature 515.7525(2014): 134-137; Gabrilovich et al, nat. reviews immunol.12.4(2012): 253-68; and Kim et al, proc.acad.sci.u.s.a.111.32(2014): 1-6). These results show that MDSCs can be targeted with CART33 alone or in combination with other immunotherapies.

Example 8: stimulation of CART proliferation by low dose RAD001 in cell culture models

The effect of low doses of RAD001 on CAR T cell proliferation in vitro was assessed by co-culturing CART-expressing cells with target cells in the presence of different concentrations of RAD 001.

Materials and methods

Generating CAR-transduced T cells

Humanized anti-human CD19 CAR (huCART19) lentiviral transfer vectors were used to generate genomic material that was packaged into VSVg-pseudotyped lentiviral particles. The amino acid and nucleotide sequences of a humanized anti-human CD19 CAR (huCART19) are CAR1, ID104875 described in PCT publication WO 2014/153270 filed 3/15 2014, and are referred to herein as SEQ ID NOs: 85 and 31.

Lenti-X293T cells were transfected by mixing lentivirus transfer vector DNA with three packaging components, VSVg env, gag/pol and rev, along with lipofectamine reagents. The medium was changed after 24 and 30 hours, after which the virus-containing medium was collected, filtered and stored at-80 ℃. CART was generated by transducing fresh or frozen naive T cells obtained by negative magnetic selection of healthy donor blood or Leukopak. T cells were activated by incubation with anti-CD 3/anti-CD 28 beads for 24 hours, after which viral supernatant or concentrated virus (

MOI

2 or 10, respectively) was added to the culture. The modified T cells were allowed to expand for about 10 days. Between day 7 and day 9, the percentage of transduced cells (expressing CAR on the cell surface) and CAR expression levels (relative fluorescence intensity, geometric mean) were determined by flow cytometry analysis. The combination of the reduced growth rate and the T cell size approaching about 350fL determines the status of the T cells that are to be frozen for later analysis.

Evaluation of proliferation of CART

To evaluate CART function, T cells were thawed and counted, and viability was assessed by means of a cell counter. The number of CAR-positive cells in each culture was normalized using untransduced T cells (UTD). Titration of RAD001 (starting at 50nM) was used to examine the effect of RAD001 on CART. The target cell line used in ALL co-culture experiments was Nalm-6, a human pre-B cell Acute Lymphoblastic Leukemia (ALL) cell line expressing CD19 and transduced to express luciferase.

To measure the proliferation of CART, T cells were cultured with target cells in a ratio of 1: 1. The assay was performed for 4 days when cells stained for CD3, CD4, CD8, and CAR expression. T cell numbers were assessed by flow cytometry using counting beads as a reference.

Results

The proliferative capacity of CART cells was tested in a 4 day co-culture assay. After CAR-transduced and non-transduced T cells were cultured with Nalm-6, CAR-positive CD 3-positive T cell numbers (dark bars) and total CD 3-positive T cell numbers (light bars) were assessed (fig. 54). The huCART19 cells expanded when cultured in the presence of less than 0.016nM RAD001, and to a lesser extent at higher concentrations of the compound. Importantly, proliferation was higher at both 0.0032nM and 0.016nM RAD001 than that observed without RAD001 addition. Untransduced T cells (UTD) showed no detectable expansion.

Example 9: low dose RAD001 stimulation of CART in vivo expansion

This example evaluated the ability of huCAR19 cells to proliferate in vivo at different concentrations of RAD 001.

The material and the method are as follows:

NALM6-luc cells: NALM6 human Acute Lymphoblastic Leukemia (ALL) cell line was developed from peripheral blood of relapsed ALL patients. The cells were then tagged with firefly luciferase. These suspension cells were grown in RPMI supplemented with 10% heat-inactivated fetal bovine serum.

Mice: receiving 6-week-old NSG (NOD. Cg-Prkdc) from Jackson Laboratory^scidIl2rg^tm1Wjl/SzJ) mice (stock No. 005557).

Tumor implantation: NALM6-luc cells were grown and expanded in vitro in RPMI supplemented with 10% heat-inactivated fetal bovine serum. The cells were then transferred to a 15ml conical tube and washed twice with cold sterile PBS. NALM6-luc cells were then counted and counted at 10X10⁶Individual cells/ml PBS concentration were resuspended. Cells were placed on ice and immediately (within 1 hour) implanted into mice. NALM6-luc cells were injected intravenously via tail vein in a volume of 100. mu.l for a total of 1X10⁶Individual cells/mouse.

CAR T cell dosing: mice were administered 5x10 7 days after tumor implantation⁶And (c) a CAR T cell. Cells were partially thawed in a 37 ℃ water bath and then completely thawed by adding 1ml of cold sterile PBS to the tube containing the cells. Thawed cells were transferred to 15ml falcon tubes and adjusted to a final volume of 10ml with PBS. Cells were washed 2 times at 1000rpm for 10 minutes each and then counted on a hemocytometer plate. T cells were then plated at 50x10⁶Individual CAR T cell/ml concentration of cold PBS resuspended and kept on ice until administrationA mouse. Mice were injected intravenously with 100 μ l CAR T cells via tail vein at a dose of 5x10 per mouse ⁶And (c) a CAR T cell. 8 mice/group were treated with 100 μ l pbs (pbs) only or humanized CD19 CAR T cells.

Administration of RAD 001: 50mg of a concentrated microemulsion equivalent to 1mg of RAD001 was formulated and then resuspended in D5W (dextrose 5% in water) at the time of administration. Mice were dosed orally (by oral gavage) daily with 200 μ l of the required dose of RAD 001.

PK analysis: mice were dosed daily with RAD001 starting 7 days after tumor implantation. The administration groups were as follows: 0.3mg/kg, 1mg/kg, 3mg/kg and 10 mg/kg. Mice were assayed for PK by bleeding after the first and last dose of RAD001 on

days

0 and 14 at the following time points: 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours.

As a result:

expansion and pharmacokinetics of RAD001 were detected in NSG mice bearing NALM6-luc tumors. Daily oral administration of RAD001 alone did not affect NALM6-luc tumor growth (fig. 55). Pharmacokinetic analysis of RAD001 showed it to be fairly stable in the blood of tumor-bearing mice (fig. 56A and 56B). PK analyses on both day 0 and day 14 showed that the blood concentration of RAD001 was above 10nm when administered at the lowest dose tested (0.3mg/kg), even 24 hours after administration.

Based on these doses, huCAR19 CAR T cells were dosed with or without RAD001 to determine the proliferative capacity of these cells. The highest dose used was 3mg/kg based on the level of RAD001 in the blood 24 hours after dosing. Since RAD001 concentrations were above 10nM 24 hours after end dose RAD001, several lower doses of RAD001 were used in the in vivo study of CAR T cells. CAR T cells were administered intravenously 1 day before initiation of daily oral dosing of RAD 001. The expansion of T cells in mice was monitored by FACS.

The lowest dose of RAD001 showed enhanced CAR T cell proliferation (figure 57). Using CD4⁺This enhanced proliferation ratio, CD8, when CAR T cells⁺CAR T cells were more pronounced and prolonged. However, CD8 was used⁺When the CAR T cells are administered, they can be administered at an early time point after administration of the CAR T cellsTo enhanced proliferation.

Example 10: in vivo anti-tumor activity of CD33 CAR-transduced T cells

HL-60 is a human acute promyelocytic leukemia cell line isolated from peripheral blood of an AML patient of the 36-year-old caucasian female species and can be grown as a xenograft in immunocompromised mice. This xenograft mimics the disease seen in bone marrow as in humans, creating a model by which the efficacy of each therapy on AML in bone can be tested. These mice can be used to test the efficacy of Chimeric Antigen Receptor (CAR) T cells specific for a cellular marker present on acute myeloid (or promyelocytic) leukemia cells, such as CD33 (Siglec-3).

Cg-Prkdc for Acute Myeloid Leukemia (AML) using HL-60 cells tagged with firefly luciferase reporter^scidIl2rg^tm1Wjl/szj (nsg) mouse orthotopic model to test the efficacy of CAR T cells specific for CD 33.

CD33 expression was detected on HL-60 cells, and these cells were used in vitro assays to observe the ability of CD33 specific CAR T cells to recognize and respond to targets. In vivo, HL-60 cells grew when implanted intravenously via the tail vein and were primarily restricted to bone marrow growth. 1 week after tumor cell implantation, the disease was well metastasized to bone and began to grow at an exponential rate. Without treatment, mice will show clinical symptoms and hind limb paralysis starting 4-6 weeks after tumor implantation. Several CD33scFv clones from in vitro screens were tested in this in vivo model.

The material and the method are as follows:

HL-60 cell line: the HL-60 human AML cell line was developed from peripheral blood of patients with acute promyelocytic leukemia. The cells were then tagged with firefly luciferase. These suspension cells were grown in RPMI supplemented with 10% heat-inactivated fetal bovine serum.

Tumor implantation: HL-60-luc cells in RPMI supplemented with 10% heat-inactivated fetal bovine serumAnd (4) growing and expanding. The cells were then transferred to 50ml conical tubes and washed twice with cold sterile PBS. HL-60-luc cells were then counted and counted at 10X10⁶Individual cells/ml PBS concentration were resuspended. Cells were placed on ice and immediately (within 1 hour) implanted into mice. HL-60-luc cells were injected intravenously via tail vein in a volume of 100. mu.l for a total of 1X10⁶Individual cells/mouse.

CAR T cell dosing: mice were administered 5x10 14 days after tumor implantation⁶A CAR⁺T cells. Implanted cells grew slowly in mice; by day 14, they have begun to increase tumor growth rate. Cells were partially thawed in a 37 ℃ water bath and then completely thawed by adding 1ml of cold sterile PBS to the tube containing the cells. Thawed cells were transferred to 15ml falcon tubes and adjusted to a final volume of 10ml with PBS. Cells were washed 2 times at 1000rpm for 10 minutes each and then counted on a hemocytometer plate. CAR T cells were normalized to CAR transduction so that each group had the same CAR ⁺Percentage of T cells. Then 5x10⁶A CAR⁺T cells at 50x10⁶A CAR⁺The concentration of T cells/ml cold PBS was resuspended and kept on ice until mice were administered. Mice were injected intravenously with 100 μ l CAR T cells via tail vein at a dose of 5x10 per mouse⁶A CAR⁺T cells.

Each group of eight mice was treated with 100 μ l of pbs (pbs), CD19-CAR control T cells (CD19), CD33-1 (clone 1) CAR T cells, CD33-2 (clone 2) CAR T cells, CD33-4 (clone 4) CAR T cells, CD33-5 (clone 5) CAR T cells, CD33-6 (clone 6) CAR T cells, CD33-7 (clone 7) CAR T cells, and CD33-9 (clone 9) CAR T cells alone. T cells were all prepared in parallel from the same human donor.

Animal monitoring: the health status of the mice was monitored daily, including body weight measurements twice weekly. Percent weight change was calculated as (BW)_{At present, the current}–BW_{Initially of})/(BW_{Initially of}) x 100%. Tumor burden was monitored twice weekly by bioluminescence imaging. Mice were injected intraperitoneally with D-luciferin and after 10 minutes the mice were anesthetized and imaged with Xenogen. By counting tumoursBioluminescence (photons/sec) of the cells, calculating the disease burden.

As a result:

the anti-tumor activity of 7 CD33 CARs was evaluated in a human Acute Myeloid Leukemia (AML) HL-60 model and compared directly to CAR T cells against CD 19. After tumor implantation on day 0, mice were randomized into treatment groups and treated with 5x10 on day 7 ⁶A CAR⁺T cell intravenous therapy. AML disease burden and animal health were monitored until the animals achieved the endpoint. On day 22 after CAR T cell administration (29 days post tumor implantation), mice in the control PBS group and CD19 CAR T cell group were euthanized when the disease burden in the control group was nearly maximal luminescence by imaging. On day 29 after CAR T cell administration (36 days post tumor implantation), mice in these groups were euthanized when the disease burden in the CD33-1, CD33-2, CD33-7, and CD33-9 CAR T cell therapy groups had reached luminescence that euthanized the control group. Mice in the CD33-4, CD33-5, and CD33-6CAR T cell therapy groups showed a reduction in late disease burden.

The bioluminescence imaging results of this study are shown in figure 58. Mean bioluminescence (+/-SEM) of tumor cells shows disease burden in intact animals. This is shown as photons/sec (p/s) of the ROI (region of interest), which is the entire mouse. PBS treated groups that did not receive any T cells demonstrated baseline HL-60 tumor growth kinetics in intravenously implanted NSG mice. The CD19 treated group received control CAR T cells, not specific for HL-60 cells, that underwent the same in vitro expansion process as CD33 CAR T cells. These cells served as T cell controls in this tumor model to show non-specific responses of T cells. Both the PBS-treated group and the CD19 CAR T cell-treated group showed continued tumor progression throughout the experiment. At injection 5X10 ⁶After each CAR T cell, all CD33 CAR T cells delayed disease progression, although these clones appeared to be divided into two differentially-responsive groups.

In this efficacy study, the anti-tumor activity of CD33 CAR-transduced T cells was evaluated in an NSG mouse model carrying human AML xenografts. These studies showed that the HL-60-luc model recapitulates human AML and can be targeted by CD33 CAR T cells in NSG mice (figure 58). Growth of HL-60-luc human AML xenografts in NSG mice after treatment with CD 33-specific CAR T cells showed a delay in disease progression (figure 58). This study showed that 7 CD33 CARs were able to exert an anti-tumor response in AML xenograft models (fig. 58).

Equivalents of

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to specific aspects, it is apparent that other aspects and modifications of the invention can be devised by others skilled in the art without departing from the true spirit and scope of the invention. It is intended that the following claims be interpreted to embrace all such aspects and their equivalents.

Sequence listing

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Binsylvania university Hospital

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gatgcccatt tgacttggga ggttgccgga aaggtaccca cagggggggt tgaggaaggg 360

ttgctggagc gccattccaa tggctctcag agccagcact caagactcac ccttccgaga 420

tccctgtgga acgccgggac ctctgtcaca tgtactctaa atcatcctag cctgccccca 480

cagcgtctga tggcccttag agagccagcc gcccaggcac cagttaagct tagcctgaat 540

ctgctcgcca gtagtgatcc cccagaggcc gccagctggc tcttatgcga agtgtccggc 600

tttagcccgc ccaacatctt gctcatgtgg ctggaggacc agcgagaagt gaacaccagc 660

ggcttcgctc cagcccggcc cccaccccag ccgggttcta ccacattctg ggcctggagt 720

gtcttaaggg tcccagcacc acctagcccc cagccagcca catacacctg tgttgtgtcc 780

catgaagata gcaggaccct gctaaatgct tctaggagtc tggaggtttc ctacgtgact 840

gaccatt 847

<210> 16

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of Artificial sequence-synthetic oligonucleotides"

<400> 16

ggtggcggag gttctggagg tggaggttcc 30

<210> 17

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of Artificial sequence-synthetic oligonucleotides"

<400> 17

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 18

<211> 126

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<400> 18

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 19

<211> 123

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<400> 19

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 20

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<400> 20

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 21

<211> 336

<212> DNA

<213> human

<400> 21

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 22

<211> 373

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence: synthetic polypeptide"

<400> 22

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

145 150 155 160

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

165 170 175

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

180 185 190

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

195 200 205

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

210 215 220

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

225 230 235 240

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

245 250 255

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

260 265 270

Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

275 280 285

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

290 295 300

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

305 310 315 320

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

325 330 335

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

340 345 350

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

355 360 365

Ala Leu Pro Pro Arg

370

<210> 23

<211> 1182

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<400> 23

atggccctcc ctgtcactgc cctgcttctc cccctcgcac tcctgctcca cgccgctaga 60

ccacccggat ggtttctgga ctctccggat cgcccgtgga atcccccaac cttctcaccg 120

gcactcttgg ttgtgactga gggcgataat gcgaccttca cgtgctcgtt ctccaacacc 180

tccgaatcat tcgtgctgaa ctggtaccgc atgagcccgt caaaccagac cgacaagctc 240

gccgcgtttc cggaagatcg gtcgcaaccg ggacaggatt gtcggttccg cgtgactcaa 300

ctgccgaatg gcagagactt ccacatgagc gtggtccgcg ctaggcgaaa cgactccggg 360

acctacctgt gcggagccat ctcgctggcg cctaaggccc aaatcaaaga gagcttgagg 420

gccgaactga gagtgaccga gcgcagagct gaggtgccaa ctgcacatcc atccccatcg 480

cctcggcctg cggggcagtt tcagaccctg gtcacgacca ctccggcgcc gcgcccaccg 540

actccggccc caactatcgc gagccagccc ctgtcgctga ggccggaagc atgccgccct 600

gccgccggag gtgctgtgca tacccgggga ttggacttcg catgcgacat ctacatttgg 660

gctcctctcg ccggaacttg tggcgtgctc cttctgtccc tggtcatcac cctgtactgc 720

aagcggggtc ggaaaaagct tctgtacatt ttcaagcagc ccttcatgag gcccgtgcaa 780

accacccagg aggaggacgg ttgctcctgc cggttccccg aagaggaaga aggaggttgc 840

gagctgcgcg tgaagttctc ccggagcgcc gacgcccccg cctataagca gggccagaac 900

cagctgtaca acgaactgaa cctgggacgg cgggaagagt acgatgtgct ggacaagcgg 960

cgcggccggg accccgaaat gggcgggaag cctagaagaa agaaccctca ggaaggcctg 1020

tataacgagc tgcagaagga caagatggcc gaggcctact ccgaaattgg gatgaaggga 1080

gagcggcgga ggggaaaggg gcacgacggc ctgtaccaag gactgtccac cgccaccaag 1140

gacacatacg atgccctgca catgcaggcc cttccccctc gc 1182

<210> 24

<211> 394

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence: synthetic polypeptide"

<400> 24

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro

20 25 30

Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly

35 40 45

Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe

50 55 60

Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu

65 70 75 80

Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe

85 90 95

Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val

100 105 110

Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser

115 120 125

Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg

130 135 140

Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser

145 150 155 160

Pro Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Thr Thr Thr Pro Ala

165 170 175

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

180 185 190

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

195 200 205

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

210 215 220

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

225 230 235 240

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

245 250 255

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

260 265 270

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

275 280 285

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

290 295 300

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

305 310 315 320

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

325 330 335

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

340 345 350

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

355 360 365

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

370 375 380

Ala Leu His Met Gln Ala Leu Pro Pro Arg

385 390

<210> 25

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 25

Gly Gly Gly Gly Ser

1 5

<210> 26

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence: synthetic polypeptide"

<220>

<221> misc_feature

<222> (1)..(30)

<223 >/Note = "this sequence may contain 1-6 'Gly Gly Gly Gly Ser' repeat units, where some sites may not be present"

<220>

<221> sources

<223 >/Note = "see alternative and preferred embodiments detailed in the specification submitted"

<400> 26

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 27

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 27

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 28

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 28

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 29

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 29

Gly Gly Gly Ser

1

<210> 30

<211> 5000

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<220>

<221> misc_feature

<222> (1)..(5000)

<223 >/Note = "this sequence may contain 50-5,000 nucleotides, where some sites may not be present"

<220>

<221> sources

<400> 30

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2040

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2100

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2160

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2220

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2280

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2340

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2400

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2460

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2520

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2580

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2640

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2700

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2760

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2820

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2880

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2940

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3000

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3060

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4980

aaaaaaaaaa aaaaaaaaaa 5000

<210> 31

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<400> 31

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60

tttttttttt tttttttttt tttttttttt tttttttttt 100

<210> 32

<211> 5000

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<220>

<221> misc_feature

<222> (1)..(5000)

<220>

<221> sources

<400> 32

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 180

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 240

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 300

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 360

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 420

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 480

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 540

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 600

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 660

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 720

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 780

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 840

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 900

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 960

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1020

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1080

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1140

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1200

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1260

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1320

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1380

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1440

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1500

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1560

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1620

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1680

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1740

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1800

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1860

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1920

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1980

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2040

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2100

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2160

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2220

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2280

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2340

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2400

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2460

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2520

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2580

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2640

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2700

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2760

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2820

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2880

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2940

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3000

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3060

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3120

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3180

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3240

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3300

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3360

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3420

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3480

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3540

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3600

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3660

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3720

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3780

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3840

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3900

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3960

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4020

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4080

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4140

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4200

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4260

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4320

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4380

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4440

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4500

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4560

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4620

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4680

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4740

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4800

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4860

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4920

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4980

tttttttttt tttttttttt 5000

<210> 33

<211> 5000

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<220>

<221> misc_feature

<222> (1)..(5000)

<223 >/Note = "this sequence may contain 100-5,000 nucleotides, where some sites may not be present"

<220>

<221> sources

<400> 33

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2040

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2100

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2160

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2220

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2280

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2340

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2400

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2460

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2520

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2580

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2640

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2700

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2760

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2820

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2880

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2940

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3000

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3060

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4980

aaaaaaaaaa aaaaaaaaaa 5000

<210> 34

<211> 400

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<220>

<221> misc_feature

<222> (1)..(400)

<223 >/Note = "this sequence may contain 100-400 nucleotides, where some sites may not be present"

<220>

<221> sources

<400> 34

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 400

<210> 35

<211> 2000

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<220>

<221> misc_feature

<222> (1)..(2000)

<223 >/Note = "this sequence may contain 50-2,000 nucleotides,

in which some sites may not be present "

<220>

<221> sources

<400> 35

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980

aaaaaaaaaa aaaaaaaaaa 2000

<210> 36

<211> 230

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence: synthetic polypeptide"

<400> 36

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys Met

225 230

<210> 37

<211> 690

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<400> 37

gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc 60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag 120

gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt caactggtac 180

gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gttcaatagc 240

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa 300

tacaagtgta aggtgtccaa caagggcctg cccagcagca tcgagaaaac catcagcaag 360

gccaagggcc agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg 420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 480

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 540

gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag 600

gagggcaacg tctttagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagagcctga gcctgtccct gggcaagatg 690

<210> 38

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence: synthetic polypeptide"

<220>

<221> misc_feature

<222> (1)..(40)

<223 >/Note = "this sequence may contain 1-10 'Gly Gly Gly Ser' repeat units, where some sites may not be present"

<220>

<221> sources

<400> 38

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

20 25 30

Gly Gly Gly Ser Gly Gly Gly Ser

35 40

<210> 39

<211> 271

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence: synthetic polypeptide"

<400> 39

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Arg Leu Gly Gly Ser Leu Pro Asp Tyr Gly

115 120 125

Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

145 150 155 160

Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu

165 170 175

Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn

180 185 190

Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

195 200 205

Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp

210 215 220

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

225 230 235 240

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu

245 250 255

Gln Thr Leu Ile Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

260 265 270

<210> 40

<211> 272

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 40

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Ala Ser Val Arg Val Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Ile Phe Thr Asn Tyr Tyr Val His Trp Val Arg Gln Ala Pro Gly Gln

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Ser Pro Ser Gly Gly Ser Pro Thr

65 70 75 80

Tyr Ala Gln Arg Leu Gln Gly Arg Val Thr Met Thr Arg Asp Leu Ser

85 90 95

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr

100 105 110

Ala Val Tyr Phe Cys Ala Arg Glu Ser Arg Leu Arg Gly Asn Arg Leu

115 120 125

Gly Leu Gln Ser Ser Ile Phe Asp His Trp Gly Gln Gly Thr Leu Val

130 135 140

Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Gly Gly Gly Gly Ser Asp Ile Arg Met Thr Gln Ser Pro Pro Ser Leu

165 170 175

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Pro Cys Gln Ala Ser Gln

180 185 190

Asp Ile Asn Asn His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala

195 200 205

Pro Gln Leu Leu Ile Tyr Asp Thr Ser Asn Leu Glu Ile Gly Val Pro

210 215 220

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

225 230 235 240

Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr

245 250 255

Glu Asn Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

260 265 270

<210> 41

<211> 270

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 41

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Lys Glu Asp Thr Ile Arg Gly Pro Asn Tyr

115 120 125

Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

130 135 140

Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Glu Thr Thr Leu Thr Gln Ser Pro Ser Ser Val Ser Ala

165 170 175

Ser Val Gly Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Asp Ile

180 185 190

Asp Thr Trp Leu Ala Trp Tyr Gln Leu Lys Pro Gly Lys Ala Pro Lys

195 200 205

Leu Leu Met Tyr Ala Ala Ser Asn Leu Gln Gly Gly Val Pro Ser Arg

210 215 220

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ile Leu Thr Ile Ser Ser

225 230 235 240

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Ser Ile

245 250 255

Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys

260 265 270

<210> 42

<211> 271

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 42

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Thr Thr Ala Tyr Leu Gln Trp Ser Ser Leu Arg Ala Ser Asp Ser

100 105 110

Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Ser Asp Tyr Asp Tyr Tyr

115 120 125

Phe Asp Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

145 150 155 160

Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu

165 170 175

Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn

180 185 190

Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

195 200 205

Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp

210 215 220

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

225 230 235 240

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu

245 250 255

Gln Thr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

260 265 270

<210> 43

<211> 261

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 43

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Asp Leu

20 25 30

Ala Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Val Ile Trp Pro Asp Gly Gly Gln Lys Tyr

65 70 75 80

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Pro

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Ile Tyr Tyr Cys Val Arg His Phe Asn Ala Trp Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln

145 150 155 160

Ser Pro Ser Ser Leu Ser Ala Tyr Val Gly Gly Arg Val Thr Ile Thr

165 170 175

Cys Gln Ala Ser Gln Gly Ile Ser Gln Phe Leu Asn Trp Phe Gln Gln

180 185 190

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Ser Asp Ala Ser Asn Leu

195 200 205

Glu Pro Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

210 215 220

Phe Thr Phe Thr Ile Thr Asn Leu Gln Pro Glu Asp Ile Ala Thr Tyr

225 230 235 240

Tyr Cys Gln Gln Tyr Asp Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr

245 250 255

Lys Val Glu Ile Lys

260

<210> 44

<211> 269

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence: synthetic polypeptide"

<400> 44

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val

20 25 30

Val Gln Pro Gly Lys Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ile Phe Ala Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Thr Ile Ser Tyr Asp Gly Ser Asn Ala Phe

65 70 75 80

Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asp Ser Leu Tyr Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Val Lys Ala Gly Asp Gly Gly Tyr Asp Val Phe

115 120 125

Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile

145 150 155 160

Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro

165 170 175

Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly

180 185 190

Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln

195 200 205

Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp Arg

210 215 220

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg

225 230 235 240

Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu Gln

245 250 255

Thr Pro Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

260 265

<210> 45

<211> 266

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 45

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Lys Glu Thr Asp Tyr Tyr Gly Ser Gly Thr

115 120 125

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

145 150 155 160

Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

165 170 175

Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Gly Ile Gly Ile Tyr Leu

180 185 190

Ala Trp Tyr Gln Gln Arg Ser Gly Lys Pro Pro Gln Leu Leu Ile His

195 200 205

Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

210 215 220

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

225 230 235 240

Asp Phe Ala Ser Tyr Trp Cys Gln Gln Ser Asn Asn Phe Pro Pro Thr

245 250 255

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

260 265

<210> 46

<211> 266

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 46

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Ala Ser Val Arg Val Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Met Phe Thr Asp Phe Phe Ile His Trp Val Arg Gln Ala Pro Gly Gln

50 55 60

Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Val Thr Lys

65 70 75 80

Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser

85 90 95

Ile Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Thr Trp Tyr Ser Ser Gly Trp Tyr Gly Ile

115 120 125

Ala Asn Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

145 150 155 160

Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

165 170 175

Arg Val Thr Ile Thr Cys Gln Ala Ser His Asp Ile Ser Asn Tyr Leu

180 185 190

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

195 200 205

Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Thr Gly Ser

210 215 220

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro Glu

225 230 235 240

Asp Val Ala Ala Tyr Tyr Cys Gln Gln Ser Asp Asp Leu Pro His Thr

245 250 255

Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

260 265

<210> 47

<211> 265

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 47

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Asn Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Arg His Gly Pro Ser Ser Trp Gly Glu Phe

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile

145 150 155 160

Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg

165 170 175

Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

180 185 190

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala

195 200 205

Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

210 215 220

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

225 230 235 240

Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe

245 250 255

Gly Gly Gly Thr Lys Val Asp Ile Lys

260 265

<210> 48

<211> 494

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 48

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Arg Leu Gly Gly Ser Leu Pro Asp Tyr Gly

115 120 125

Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

145 150 155 160

Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu

165 170 175

Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn

180 185 190

Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

195 200 205

Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp

210 215 220

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

225 230 235 240

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu

245 250 255

Gln Thr Leu Ile Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Thr

260 265 270

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

275 280 285

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

290 295 300

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

305 310 315 320

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

325 330 335

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

340 345 350

Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

355 360 365

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val

370 375 380

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn

385 390 395 400

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

405 410 415

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

420 425 430

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

435 440 445

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

450 455 460

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

465 470 475 480

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 49

<211> 495

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 49

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Ala Ser Val Arg Val Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Ile Phe Thr Asn Tyr Tyr Val His Trp Val Arg Gln Ala Pro Gly Gln

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Ser Pro Ser Gly Gly Ser Pro Thr

65 70 75 80

Tyr Ala Gln Arg Leu Gln Gly Arg Val Thr Met Thr Arg Asp Leu Ser

85 90 95

Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr

100 105 110

Ala Val Tyr Phe Cys Ala Arg Glu Ser Arg Leu Arg Gly Asn Arg Leu

115 120 125

Gly Leu Gln Ser Ser Ile Phe Asp His Trp Gly Gln Gly Thr Leu Val

130 135 140

Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Gly Gly Gly Gly Ser Asp Ile Arg Met Thr Gln Ser Pro Pro Ser Leu

165 170 175

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Pro Cys Gln Ala Ser Gln

180 185 190

Asp Ile Asn Asn His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala

195 200 205

Pro Gln Leu Leu Ile Tyr Asp Thr Ser Asn Leu Glu Ile Gly Val Pro

210 215 220

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

225 230 235 240

Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr

245 250 255

Glu Asn Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

260 265 270

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

275 280 285

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

290 295 300

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

305 310 315 320

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

325 330 335

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

340 345 350

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

355 360 365

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

435 440 445

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

450 455 460

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

465 470 475 480

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 50

<211> 493

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 50

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Lys Glu Asp Thr Ile Arg Gly Pro Asn Tyr

115 120 125

Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

130 135 140

Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Glu Thr Thr Leu Thr Gln Ser Pro Ser Ser Val Ser Ala

165 170 175

Ser Val Gly Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Asp Ile

180 185 190

Asp Thr Trp Leu Ala Trp Tyr Gln Leu Lys Pro Gly Lys Ala Pro Lys

195 200 205

Leu Leu Met Tyr Ala Ala Ser Asn Leu Gln Gly Gly Val Pro Ser Arg

210 215 220

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ile Leu Thr Ile Ser Ser

225 230 235 240

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Ser Ile

245 250 255

Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Thr Thr

260 265 270

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

275 280 285

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

290 295 300

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

305 310 315 320

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

325 330 335

Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

340 345 350

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

355 360 365

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

370 375 380

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln

385 390 395 400

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

405 410 415

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

420 425 430

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

435 440 445

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

450 455 460

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

465 470 475 480

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 51

<211> 494

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 51

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Thr Thr Ala Tyr Leu Gln Trp Ser Ser Leu Arg Ala Ser Asp Ser

100 105 110

Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Ser Asp Tyr Asp Tyr Tyr

115 120 125

Phe Asp Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

145 150 155 160

Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu

165 170 175

Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn

180 185 190

Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

195 200 205

Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp

210 215 220

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

225 230 235 240

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu

245 250 255

Gln Thr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr

260 265 270

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

275 280 285

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

290 295 300

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

305 310 315 320

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

325 330 335

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

340 345 350

Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

355 360 365

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val

370 375 380

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn

385 390 395 400

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

405 410 415

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

420 425 430

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

435 440 445

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

450 455 460

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

465 470 475 480

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 52

<211> 484

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 52

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Asp Leu

20 25 30

Ala Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Val Ile Trp Pro Asp Gly Gly Gln Lys Tyr

65 70 75 80

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Pro

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Ile Tyr Tyr Cys Val Arg His Phe Asn Ala Trp Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln

145 150 155 160

Ser Pro Ser Ser Leu Ser Ala Tyr Val Gly Gly Arg Val Thr Ile Thr

165 170 175

Cys Gln Ala Ser Gln Gly Ile Ser Gln Phe Leu Asn Trp Phe Gln Gln

180 185 190

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Ser Asp Ala Ser Asn Leu

195 200 205

Glu Pro Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

210 215 220

Phe Thr Phe Thr Ile Thr Asn Leu Gln Pro Glu Asp Ile Ala Thr Tyr

225 230 235 240

Tyr Cys Gln Gln Tyr Asp Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr

245 250 255

Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

260 265 270

Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

275 280 285

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

290 295 300

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu

305 310 315 320

Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys

325 330 335

Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr

340 345 350

Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly

355 360 365

Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

370 375 380

Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

385 390 395 400

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

405 410 415

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

420 425 430

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

435 440 445

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

450 455 460

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

465 470 475 480

Leu Pro Pro Arg

<210> 53

<211> 492

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 53

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val

20 25 30

Val Gln Pro Gly Lys Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ile Phe Ala Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Thr Ile Ser Tyr Asp Gly Ser Asn Ala Phe

65 70 75 80

Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asp Ser Leu Tyr Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Val Lys Ala Gly Asp Gly Gly Tyr Asp Val Phe

115 120 125

Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile

145 150 155 160

Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro

165 170 175

Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly

180 185 190

Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln

195 200 205

Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp Arg

210 215 220

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg

225 230 235 240

Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu Gln

245 250 255

Thr Pro Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Thr Thr Thr

260 265 270

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

275 280 285

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

290 295 300

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

305 310 315 320

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

325 330 335

Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

340 345 350

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

355 360 365

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

370 375 380

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu

385 390 395 400

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

405 410 415

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

420 425 430

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

435 440 445

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

450 455 460

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

465 470 475 480

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 54

<211> 489

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 54

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Lys Glu Thr Asp Tyr Tyr Gly Ser Gly Thr

115 120 125

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

145 150 155 160

Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

165 170 175

Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Gly Ile Gly Ile Tyr Leu

180 185 190

Ala Trp Tyr Gln Gln Arg Ser Gly Lys Pro Pro Gln Leu Leu Ile His

195 200 205

Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

210 215 220

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

225 230 235 240

Asp Phe Ala Ser Tyr Trp Cys Gln Gln Ser Asn Asn Phe Pro Pro Thr

245 250 255

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro

260 265 270

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

275 280 285

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

290 295 300

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

305 310 315 320

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys

325 330 335

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

340 345 350

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro

355 360 365

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

370 375 380

Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu

385 390 395 400

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

420 425 430

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

435 440 445

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

450 455 460

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg

485

<210> 55

<211> 489

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 55

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Ala Ser Val Arg Val Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Met Phe Thr Asp Phe Phe Ile His Trp Val Arg Gln Ala Pro Gly Gln

50 55 60

Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Val Thr Lys

65 70 75 80

Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser

85 90 95

Ile Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Thr Trp Tyr Ser Ser Gly Trp Tyr Gly Ile

115 120 125

Ala Asn Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

145 150 155 160

Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

165 170 175

Arg Val Thr Ile Thr Cys Gln Ala Ser His Asp Ile Ser Asn Tyr Leu

180 185 190

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

195 200 205

Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Thr Gly Ser

210 215 220

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro Glu

225 230 235 240

Asp Val Ala Ala Tyr Tyr Cys Gln Gln Ser Asp Asp Leu Pro His Thr

245 250 255

Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Thr Thr Thr Pro Ala Pro

260 265 270

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

275 280 285

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

290 295 300

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

305 310 315 320

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys

325 330 335

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

340 345 350

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro

355 360 365

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

370 375 380

Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu

385 390 395 400

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

420 425 430

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

435 440 445

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

450 455 460

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg

485

<210> 56

<211> 488

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 56

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Asn Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Arg His Gly Pro Ser Ser Trp Gly Glu Phe

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile

145 150 155 160

Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg

165 170 175

Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

180 185 190

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala

195 200 205

Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

210 215 220

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

225 230 235 240

Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe

245 250 255

Gly Gly Gly Thr Lys Val Asp Ile Lys Thr Thr Thr Pro Ala Pro Arg

260 265 270

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg

325 330 335

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

340 345 350

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

355 360 365

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

385 390 395 400

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

405 410 415

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

420 425 430

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

435 440 445

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

450 455 460

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg

485

<210> 57

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 57

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Gly Ser Leu Pro Asp Tyr Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 58

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 58

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Arg Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Tyr Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Ser Pro Ser Gly Gly Ser Pro Thr Tyr Ala Gln Arg Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Leu Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Ser Arg Leu Arg Gly Asn Arg Leu Gly Leu Gln Ser Ser

100 105 110

Ile Phe Asp His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 59

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 59

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Glu Asp Thr Ile Arg Gly Pro Asn Tyr Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 60

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 60

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Thr Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Arg Ala Ser Asp Ser Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Ser Asp Tyr Asp Tyr Tyr Phe Asp Phe Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 61

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 61

Gln Val Gln Leu Val Gln Ser Gly Gly Asp Leu Ala Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Pro Asp Gly Gly Gln Lys Tyr Tyr Gly Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Pro Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Val Arg His Phe Asn Ala Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 62

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 62

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Lys

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Phe

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Tyr Asp Gly Ser Asn Ala Phe Tyr Ala Asp Ser Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asp Ser Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Ala Gly Asp Gly Gly Tyr Asp Val Phe Asp Ser Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 63

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 63

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Glu Thr Asp Tyr Tyr Gly Ser Gly Thr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 64

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 64

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Arg Val Ser Cys Lys Ala Ser Gly Tyr Met Phe Thr Asp Phe

20 25 30

Phe Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Val Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Trp Tyr Ser Ser Gly Trp Tyr Gly Ile Ala Asn Ile Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 65

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 65

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Gly Pro Ser Ser Trp Gly Glu Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 66

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 66

Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Leu Ile Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105 110

<210> 67

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 67

Asp Ile Arg Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Pro Cys Gln Ala Ser Gln Asp Ile Asn Asn His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Gln Leu Leu Ile

35 40 45

Tyr Asp Thr Ser Asn Leu Glu Ile Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Glu Asn Leu Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 68

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 68

Glu Thr Thr Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Asp Ile Asp Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Leu Lys Pro Gly Lys Ala Pro Lys Leu Leu Met

35 40 45

Tyr Ala Ala Ser Asn Leu Gln Gly Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ile Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Ser Ile Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys

100 105

<210> 69

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 69

Glu Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 70

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 70

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Tyr Val Gly

1 5 10 15

Gly Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Gly Ile Ser Gln Phe

20 25 30

Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Asp Ala Ser Asn Leu Glu Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Thr Asn Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asp Leu Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 71

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 71

Glu Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105 110

<210> 72

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 72

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Gly Ile Gly Ile Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Arg Ser Gly Lys Pro Pro Gln Leu Leu Ile

35 40 45

His Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Ser Tyr Trp Cys Gln Gln Ser Asn Asn Phe Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 73

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 73

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser His Asp Ile Ser Asn Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Ala Tyr Tyr Cys Gln Gln Ser Asp Asp Leu Pro His

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 74

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 74

Asp Ile Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys

100 105

<210> 75

<211> 1482

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 75

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccccaagtgc aactcgtcca gtccggtgca gaagtcaaga agccaggaga atcactcaag 120

attagctgca aaggcagcgg ctactccttc acttcctact ggatcggctg ggtgcgccag 180

atgcccggaa agggactgga gtggatggga atcatctacc ctggcgatag cgacaccaga 240

tactccccga gctttcaagg ccaagtgacc atttcggccg acaagtcgat ctccaccgcg 300

tatctgcagt ggagctcact gaaggcttcg gacaccgcca tgtactactg tgcccggctg 360

gggggaagcc tgcccgatta cggaatggac gtgtggggcc agggaaccat ggtcactgtg 420

tcctccgcct ccgggggtgg aggctccggt ggaggggggt ccggtggtgg aggatcagaa 480

attgtgctga cccagtctcc gctgtccttg cctgtgaccc cgggcgaacc cgcaagcatc 540

tcctgccggt cgtcgcagtc cctgcttcac tccaacggct acaactacct cgattggtac 600

ctccagaagc ctggacagag cccacagctg ttgatctacc tgggctcgaa ccgggcctca 660

ggagtgccgg acaggttctc cggctccggg tcgggaaccg acttcacgct gaagatctcc 720

cgcgtggagg ccgaggacgt gggcgtgtac tattgcatgc aggcgctgca gacccttatt 780

acattcggac aggggactaa ggtcgatatc aagaccacta ccccagcacc gaggccaccc 840

accccggctc ctaccatcgc ctcccagcct ctgtccctgc gtccggaggc atgtagaccc 900

gcagctggtg gggccgtgca tacccggggt cttgacttcg cctgcgatat ctacatttgg 960

gcccctctgg ctggtacttg cggggtcctg ctgctttcac tcgtgatcac tctttactgt 1020

aagcgcggtc ggaagaagct gctgtacatc tttaagcaac ccttcatgag gcctgtgcag 1080

actactcaag aggaggacgg ctgttcatgc cggttcccag aggaggagga aggcggctgc 1140

gaactgcgcg tgaaattcag ccgcagcgca gatgctccag cctacaagca ggggcagaac 1200

cagctctaca acgaactcaa tcttggtcgg agagaggagt acgacgtgct ggacaagcgg 1260

agaggacggg acccagaaat gggcgggaag ccgcgcagaa agaatcccca agagggcctg 1320

tacaacgagc tccaaaagga taagatggca gaagcctata gcgagattgg tatgaaaggg 1380

gaacgcagaa gaggcaaagg ccacgacgga ctgtaccagg gactcagcac cgccaccaag 1440

gacacctatg acgctcttca catgcaggcc ctgccgcctc gg 1482

<210> 76

<211> 1485

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 76

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccccaagtcc aactcgtcca atcaggagct gaagtcaaga agcctggagc atccgtgaga 120

gtgtcctgta aagcctccgg ctacatcttc accaactact acgtgcactg ggtcagacag 180

gccccgggcc agggactgga atggatggga atcatttccc cgtccggcgg atcgcctact 240

tacgcgcaac ggctgcaggg ccgcgtgacc atgactcggg atctctccac ttcaaccgtg 300

tacatggaac tgtccagcct tacatcggag gatactgccg tgtacttctg cgcgagggag 360

tcccggctga ggggcaaccg cctcgggctg cagtcaagca tcttcgatca ctggggccag 420

ggcaccctcg tgaccgtgtc cagcgcctcg gggggaggag gctccggggg cggaggttcg 480

ggcggtggtg gatctgacat tcgcatgact cagtccccac cttcactgtc cgctagcgtg 540

ggggaccgcg tgacgattcc gtgccaagcc agccaggaca tcaacaacca tctgaactgg 600

tatcagcaga agcccggaaa ggccccgcag ctgctgatct acgacacctc gaatctggag 660

atcggcgtgc catcccggtt ctccggttcg ggaagcggaa ccgactttac cctgactatc 720

tcctccttgc aacccgagga cattgccacc tactactgcc agcagtacga aaaccttccc 780

ctgaccttcg ggggtggaac caaagtggag atcaagacca ctaccccagc accgaggcca 840

cccaccccgg ctcctaccat cgcctcccag cctctgtccc tgcgtccgga ggcatgtaga 900

cccgcagctg gtggggccgt gcatacccgg ggtcttgact tcgcctgcga tatctacatt 960

tgggcccctc tggctggtac ttgcggggtc ctgctgcttt cactcgtgat cactctttac 1020

tgtaagcgcg gtcggaagaa gctgctgtac atctttaagc aacccttcat gaggcctgtg 1080

cagactactc aagaggagga cggctgttca tgccggttcc cagaggagga ggaaggcggc 1140

tgcgaactgc gcgtgaaatt cagccgcagc gcagatgctc cagcctacaa gcaggggcag 1200

aaccagctct acaacgaact caatcttggt cggagagagg agtacgacgt gctggacaag 1260

cggagaggac gggacccaga aatgggcggg aagccgcgca gaaagaatcc ccaagagggc 1320

ctgtacaacg agctccaaaa ggataagatg gcagaagcct atagcgagat tggtatgaaa 1380

ggggaacgca gaagaggcaa aggccacgac ggactgtacc agggactcag caccgccacc 1440

aaggacacct atgacgctct tcacatgcag gccctgccgc ctcgg 1485

<210> 77

<211> 1479

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 77

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccccaagtgc aattggtgca gtcaggagga ggattggtgc aacccggagg atcgctgaga 120

ctgtcatgtg ctgccagcgg gttcacattc tcctcctacg caatgtcctg ggtccgccag 180

gcgccgggca aaggactgga atgggtgtcc gccatctcgg ggtcgggcgg ctccacctat 240

tacgctgact ccgtgaaggg acgcttcacc attagcagag ataactccaa gaacaccctc 300

tacctccaaa tgaacagcct tagggctgag gacaccgccg tctattactg cgccaaggag 360

gacacgatcc ggggacctaa ctactattac tacggaatgg atgtctgggg ccagggtacc 420

actgtgaccg tgtcctcggc ctcgggaggc ggaggatcag ggggtggtgg ctctgggggg 480

ggtggcagcg aaactactct gacccagtcc ccctcatccg tgtcagcgtc cgtgggcgat 540

cgggtgtcga tcacttgccg ggcctcccaa gacatcgaca cctggctcgc gtggtaccag 600

ctgaagccag gaaaggcccc taagctgctg atgtacgcag cctccaatct gcaaggaggg 660

gtgccctccc gcttttccgg gtccggcagc ggaaccgact tcattctgac tatctcgagc 720

ctccagccgg aggatttcgc cacctactac tgccagcagg cctccatctt cccgccgact 780

ttcggtggcg gaaccaaggt cgacattaag accactaccc cagcaccgag gccacccacc 840

ccggctccta ccatcgcctc ccagcctctg tccctgcgtc cggaggcatg tagacccgca 900

gctggtgggg ccgtgcatac ccggggtctt gacttcgcct gcgatatcta catttgggcc 960

cctctggctg gtacttgcgg ggtcctgctg ctttcactcg tgatcactct ttactgtaag 1020

cgcggtcgga agaagctgct gtacatcttt aagcaaccct tcatgaggcc tgtgcagact 1080

actcaagagg aggacggctg ttcatgccgg ttcccagagg aggaggaagg cggctgcgaa 1140

ctgcgcgtga aattcagccg cagcgcagat gctccagcct acaagcaggg gcagaaccag 1200

ctctacaacg aactcaatct tggtcggaga gaggagtacg acgtgctgga caagcggaga 1260

ggacgggacc cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac 1320

aacgagctcc aaaaggataa gatggcagaa gcctatagcg agattggtat gaaaggggaa 1380

cgcagaagag gcaaaggcca cgacggactg taccagggac tcagcaccgc caccaaggac 1440

acctatgacg ctcttcacat gcaggccctg ccgcctcgg 1479

<210> 78

<211> 1482

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 78

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccccaagtgc agctcgtcca atccggtgca gaagtgaaga agcctggcga atccctgaag 120

atctcatgca aaggctcggg atacagcttc acctcatatt ggattggatg ggtcagacag 180

atgccaggaa agggtctgga gtggatggga atcatctacc cgggagacag cgatacccgg 240

tactccccga gcttccaggg acaggtcacc atctcggccg acaagtccat tactactgcc 300

tacttgcaat ggtcctcgct gcgcgcctcc gatagcgcca tgtactactg cgcgagaggc 360

ggctactccg actacgacta ctacttcgat ttctggggac aggggacact cgtgactgtg 420

tcctccgcgt cgggtggcgg cggctcgggt ggaggaggaa gcggaggggg aggctccgaa 480

attgtgatga cccagtcacc cctgtcgctc cctgtgactc ctggggaacc ggcctccatc 540

tcctgccgga gctcacagag cctgctgcac tccaacggat acaactacct cgattggtac 600

cttcagaagc ccggccagtc gccccagctg ctgatctacc tggggtccaa ccgggctagc 660

ggcgtgccgg accgcttctc cggttccggg tctggaaccg acttcacgct gaaaatctcc 720

agggtggagg ccgaggacgt gggagtgtat tactgtatgc aggccctgca aacccccttc 780

acctttggcg ggggcaccaa ggtcgagatt aagaccacta ccccagcacc gaggccaccc 840

accccggctc ctaccatcgc ctcccagcct ctgtccctgc gtccggaggc atgtagaccc 900

gcagctggtg gggccgtgca tacccggggt cttgacttcg cctgcgatat ctacatttgg 960

gcccctctgg ctggtacttg cggggtcctg ctgctttcac tcgtgatcac tctttactgt 1020

aagcgcggtc ggaagaagct gctgtacatc tttaagcaac ccttcatgag gcctgtgcag 1080

actactcaag aggaggacgg ctgttcatgc cggttcccag aggaggagga aggcggctgc 1140

gaactgcgcg tgaaattcag ccgcagcgca gatgctccag cctacaagca ggggcagaac 1200

cagctctaca acgaactcaa tcttggtcgg agagaggagt acgacgtgct ggacaagcgg 1260

agaggacggg acccagaaat gggcgggaag ccgcgcagaa agaatcccca agagggcctg 1320

tacaacgagc tccaaaagga taagatggca gaagcctata gcgagattgg tatgaaaggg 1380

gaacgcagaa gaggcaaagg ccacgacgga ctgtaccagg gactcagcac cgccaccaag 1440

gacacctatg acgctcttca catgcaggcc ctgccgcctc gg 1482

<210> 79

<211> 1452

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 79

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccccaagtgc aactcgtcca aagcggtgga gatctcgccc agcccggaag atcccttaga 120

ctctcatgtg ccgccagcgg gttcaccttc gacgactacg ctatgcattg ggtgcgccag 180

gccccgggga agggactgga atgggtggcc gtgatttggc cggacggcgg acagaagtac 240

tacggagaca gcgtgaaagg gcggttcacc gtgtcgaggg acaacccgaa gaataccctc 300

taccttcaaa tgaactccct gcgcgccgag gacaccgcga tctactactg cgtgcgccac 360

tttaacgcat gggattactg gggacagggg actctggtca ctgtgtcctc cgcctctggc 420

ggcggaggtt ccggcggtgg tggctccggt ggaggaggat cggacatcca gctgacccag 480

tccccttcct cactgtcggc gtacgtggga ggccgggtca ctatcacgtg ccaggcatcc 540

cagggcattt cccagttcct gaactggttc cagcagaagc ccggaaaggc ccctaagctg 600

ttgatttccg atgctagcaa cctggaaccc ggcgtgccgt cacggttcag cggctccggg 660

tcgggcaccg acttcacctt caccatcact aacctccaac cggaggacat cgccacctat 720

tactgccagc agtacgatga tctgccactg actttcggcg gcggaaccaa ggtcgaaatc 780

aagaccacta ccccagcacc gaggccaccc accccggctc ctaccatcgc ctcccagcct 840

ctgtccctgc gtccggaggc atgtagaccc gcagctggtg gggccgtgca tacccggggt 900

cttgacttcg cctgcgatat ctacatttgg gcccctctgg ctggtacttg cggggtcctg 960

ctgctttcac tcgtgatcac tctttactgt aagcgcggtc ggaagaagct gctgtacatc 1020

tttaagcaac ccttcatgag gcctgtgcag actactcaag aggaggacgg ctgttcatgc 1080

cggttcccag aggaggagga aggcggctgc gaactgcgcg tgaaattcag ccgcagcgca 1140

gatgctccag cctacaagca ggggcagaac cagctctaca acgaactcaa tcttggtcgg 1200

agagaggagt acgacgtgct ggacaagcgg agaggacggg acccagaaat gggcgggaag 1260

ccgcgcagaa agaatcccca agagggcctg tacaacgagc tccaaaagga taagatggca 1320

gaagcctata gcgagattgg tatgaaaggg gaacgcagaa gaggcaaagg ccacgacgga 1380

ctgtaccagg gactcagcac cgccaccaag gacacctatg acgctcttca catgcaggcc 1440

ctgccgcctc gg 1452

<210> 80

<211> 1476

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 80

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccccaagtgc aactcgtcca atccggtggt ggtgtcgtgc aaccaggaaa gtctcttcgc 120

ctctcatgcg ctgccagcgg attcacgttt tccatcttcg ctatgcactg ggtgcggcag 180

gccccgggaa agggactgga atgggtggca accatttcat acgatggatc aaacgcgttc 240

tacgccgact ccgtggaagg aaggttcacc atctcgagag acaactccaa ggactcgctg 300

tatctgcaaa tggactccct gcgccctgag gataccgccg tctactactg cgtgaaggcc 360

ggcgacgggg gatacgacgt gttcgattcg tggggccagg gaactctggt caccgtgtcc 420

agcgcgagcg ggggaggcgg atcgggtggt ggagggtccg ggggaggagg ctccgagatc 480

gtgatgactc agtcgccgct ctccctcccc gtgacccccg gagagccagc tagcatttca 540

tgtcggagct cccagtccct gctgcactcc aacggctaca attacctgga ttggtacttg 600

cagaagcctg ggcagagccc tcagctgctg atctacctcg gctcgaacag agcctccggc 660

gtgccggacc ggttttccgg gagcggcagc ggcaccgatt tcaccttgaa aatctcccgc 720

gtggaagccg aggacgtggg cgtgtactat tgcatgcagg ccctgcagac tcccaccttc 780

ggcccgggaa ctaaggtcga catcaagacc actaccccag caccgaggcc acccaccccg 840

gctcctacca tcgcctccca gcctctgtcc ctgcgtccgg aggcatgtag acccgcagct 900

ggtggggccg tgcatacccg gggtcttgac ttcgcctgcg atatctacat ttgggcccct 960

ctggctggta cttgcggggt cctgctgctt tcactcgtga tcactcttta ctgtaagcgc 1020

ggtcggaaga agctgctgta catctttaag caacccttca tgaggcctgt gcagactact 1080

caagaggagg acggctgttc atgccggttc ccagaggagg aggaaggcgg ctgcgaactg 1140

cgcgtgaaat tcagccgcag cgcagatgct ccagcctaca agcaggggca gaaccagctc 1200

tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 1260

cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 1320

gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 1380

agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 1440

tatgacgctc ttcacatgca ggccctgccg cctcgg 1476

<210> 81

<211> 1467

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 81

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

cccgaagtgc aattggtgga atctggagga ggattggtgc aacctggagg atctctgaga 120

ctgtcatgtg ccgccagcgg cttcacattt tcctcctacg cgatgtcatg ggtccgccag 180

gcaccgggga aaggactgga atgggtgtcc gccatttcgg gatcgggagg ctcgacctac 240

tacgccgaca gcgtgaaggg aagattcact atctcccggg ataactccaa gaatactctg 300

tatctccaaa tgaactccct gagggccgag gatactgccg tgtactactg cgctaaggaa 360

accgactact acggctcagg aaccttcgac tactggggcc agggcaccct cgtgaccgtg 420

tcctcggcct ccggcggcgg aggttcgggg gggggcggtt ccgggggagg gggcagcgac 480

atccagatga cccagtcccc aagctccctt tccgcgtccg tgggagatcg cgtgaccatt 540

tcgtgccggg ctagccaggg catcggtatc tatcttgcgt ggtaccagca gcggagcgga 600

aagccgcccc agctgctgat ccacggcgcc tcaactctgc aatccggggt ccccagccgg 660

ttcagcggta gcgggtcggg taccgacttt accctgacta tctcctccct ccaaccggag 720

gacttcgcct cctactggtg ccagcagtcc aacaacttcc ctcccacctt cggccaggga 780

acgaaggtcg agattaagac cactacccca gcaccgaggc cacccacccc ggctcctacc 840

atcgcctccc agcctctgtc cctgcgtccg gaggcatgta gacccgcagc tggtggggcc 900

gtgcataccc ggggtcttga cttcgcctgc gatatctaca tttgggcccc tctggctggt 960

acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag 1020

aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac tcaagaggag 1080

gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa 1140

ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct ctacaacgaa 1200

ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg acgggaccca 1260

gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa 1320

aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg cagaagaggc 1380

aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct 1440

cttcacatgc aggccctgcc gcctcgg 1467

<210> 82

<211> 1467

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 82

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccccaagtcc aactcgtcca gtccggtgca gaagtcaaga agccaggagc ctccgtgaga 120

gtgtcgtgca aagcgtccgg ctacatgttc accgactttt tcattcactg ggtgcgccag 180

gcgcccggac agggtctgga gtggatgggg tggatcaacc ctaactccgg cgtgactaaa 240

tacgcccaga agttccaggg ccgcgtgacc atgacccgga acactagcat ctccaccgcc 300

tacatggaac tgtcatccct ccggtccgag gataccgccg tgtactactg cgccacctgg 360

tacagcagcg gttggtacgg catcgcgaac atttggggac aggggactat ggtcaccgtg 420

tcatccgcct ccgggggagg agggtccggc ggcggaggtt ctggaggagg cggctcggac 480

atccagttga cgcagagccc ctcgtcgctg agcgcctccg tgggcgacag agtgaccatt 540

acctgtcaag cttcccatga tatctcgaac tacctccact ggtatcagca gaagccggga 600

aaggctccca agctgctgat ctacgacgcc tccaatctgg aaaccggagt gccgagccgg 660

ttcactggat cagggagcgg cactgacttc accctgacaa ttaggtcgct gcagccggag 720

gatgtggcag cctactactg ccaacagtca gacgaccttc ctcacacttt cggacaaggg 780

actaaggtcg acatcaagac cactacccca gcaccgaggc cacccacccc ggctcctacc 840

atcgcctccc agcctctgtc cctgcgtccg gaggcatgta gacccgcagc tggtggggcc 900

gtgcataccc ggggtcttga cttcgcctgc gatatctaca tttgggcccc tctggctggt 960

acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag 1020

aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac tcaagaggag 1080

gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa 1140

ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct ctacaacgaa 1200

ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg acgggaccca 1260

gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa 1320

aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg cagaagaggc 1380

aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct 1440

cttcacatgc aggccctgcc gcctcgg 1467

<210> 83

<211> 1464

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 83

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccccaagtgc aactcgtcca gtccggtgca gaagtgaaaa agccaggaga aagcctcaag 120

atcagctgca agggatctgg gtacagcttc accaactact ggatcggctg ggtgcgccag 180

atgcccggaa agggactgga gtggatgggc attatctacc ctggggactc cgacacccgg 240

tattccccga gcttccaagg acaggtcacc atctccgccg ataagtcgat tagcactgcg 300

tacttgcagt ggtcaagcct gaaggcctcg gacaccgcca tgtactactg cgcgagacac 360

gggccctcgt cctggggcga atttgactac tggggccagg gaacgcttgt gaccgtgtcg 420

tccgcgtccg ggggtggagg atcaggagga ggaggctccg gtggtggcgg tagcgacatc 480

cggctgactc agtccccttc ctcactctcc gcctccgtgg gggaccgcgt gaccattacc 540

tgtcgggcat cacagtccat cagctcatac ctgaactggt atcagcagaa gccggggaag 600

gccccgaaac tcctgatcta cgccgcctcc tccctgcaat ccggcgtgcc ctcgaggttc 660

tccggctccg gctcgggaac cgatttcact ctgacaatta gcagcctgca gcctgaggat 720

ttcgctacct actactgcca gcagtcctac tcgactccgc tgactttcgg cgggggaacc 780

aaggtcgaca tcaagaccac taccccagca ccgaggccac ccaccccggc tcctaccatc 840

gcctcccagc ctctgtccct gcgtccggag gcatgtagac ccgcagctgg tggggccgtg 900

catacccggg gtcttgactt cgcctgcgat atctacattt gggcccctct ggctggtact 960

tgcggggtcc tgctgctttc actcgtgatc actctttact gtaagcgcgg tcggaagaag 1020

ctgctgtaca tctttaagca acccttcatg aggcctgtgc agactactca agaggaggac 1080

ggctgttcat gccggttccc agaggaggag gaaggcggct gcgaactgcg cgtgaaattc 1140

agccgcagcg cagatgctcc agcctacaag caggggcaga accagctcta caacgaactc 1200

aatcttggtc ggagagagga gtacgacgtg ctggacaagc ggagaggacg ggacccagaa 1260

atgggcggga agccgcgcag aaagaatccc caagagggcc tgtacaacga gctccaaaag 1320

gataagatgg cagaagccta tagcgagatt ggtatgaaag gggaacgcag aagaggcaaa 1380

ggccacgacg gactgtacca gggactcagc accgccacca aggacaccta tgacgctctt 1440

cacatgcagg ccctgccgcc tcgg 1464

<210> 84

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 84

Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly

1 5 10

<210> 85

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 85

Gly Tyr Ile Phe Thr Asn Tyr Tyr Val His

1 5 10

<210> 86

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 86

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser

1 5 10

<210> 87

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 87

Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly

1 5 10

<210> 88

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 88

Gly Phe Thr Phe Asp Asp Tyr Ala Met His

1 5 10

<210> 89

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 89

Gly Phe Thr Phe Ser Ile Phe Ala Met His

1 5 10

<210> 90

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 90

Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser

1 5 10

<210> 91

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 91

Gly Tyr Met Phe Thr Asp Phe Phe Ile His

1 5 10

<210> 92

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 92

Gly Tyr Ser Phe Thr Asn Tyr Trp Ile Gly

1 5 10

<210> 93

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 93

Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 94

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 94

Ile Ile Ser Pro Ser Gly Gly Ser Pro Thr Tyr Ala Gln Arg Leu Gln

1 5 10 15

Gly

<210> 95

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 95

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 96

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 96

Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 97

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 97

Val Ile Trp Pro Asp Gly Gly Gln Lys Tyr Tyr Gly Asp Ser Val Lys

1 5 10 15

Gly

<210> 98

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 98

Thr Ile Ser Tyr Asp Gly Ser Asn Ala Phe Tyr Ala Asp Ser Val Glu

1 5 10 15

Gly

<210> 99

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 99

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 100

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 100

Trp Ile Asn Pro Asn Ser Gly Val Thr Lys Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 101

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 101

Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 102

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 102

Leu Gly Gly Ser Leu Pro Asp Tyr Gly Met Asp Val

1 5 10

<210> 103

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 103

Glu Ser Arg Leu Arg Gly Asn Arg Leu Gly Leu Gln Ser Ser Ile Phe

1 5 10 15

Asp His

<210> 104

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 104

Glu Asp Thr Ile Arg Gly Pro Asn Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 105

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 105

Gly Gly Tyr Ser Asp Tyr Asp Tyr Tyr Phe Asp Phe

1 5 10

<210> 106

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 106

His Phe Asn Ala Trp Asp Tyr

1 5

<210> 107

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 107

Ala Gly Asp Gly Gly Tyr Asp Val Phe Asp Ser

1 5 10

<210> 108

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 108

Glu Thr Asp Tyr Tyr Gly Ser Gly Thr Phe Asp Tyr

1 5 10

<210> 109

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 109

Trp Tyr Ser Ser Gly Trp Tyr Gly Ile Ala Asn Ile

1 5 10

<210> 110

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 110

His Gly Pro Ser Ser Trp Gly Glu Phe Asp Tyr

1 5 10

<210> 111

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 111

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 112

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 112

Gln Ala Ser Gln Asp Ile Asn Asn His Leu Asn

1 5 10

<210> 113

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 113

Arg Ala Ser Gln Asp Ile Asp Thr Trp Leu Ala

1 5 10

<210> 114

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 114

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 115

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 115

Gln Ala Ser Gln Gly Ile Ser Gln Phe Leu Asn

1 5 10

<210> 116

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 116

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 117

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 117

Arg Ala Ser Gln Gly Ile Gly Ile Tyr Leu Ala

1 5 10

<210> 118

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 118

Gln Ala Ser His Asp Ile Ser Asn Tyr Leu His

1 5 10

<210> 119

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 119

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 120

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 120

Leu Gly Ser Asn Arg Ala Ser

1 5

<210> 121

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 121

Asp Thr Ser Asn Leu Glu Ile

1 5

<210> 122

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 122

Ala Ala Ser Asn Leu Gln Gly

1 5

<210> 123

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 123

Leu Gly Ser Asn Arg Ala Ser

1 5

<210> 124

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 124

Asp Ala Ser Asn Leu Glu Pro

1 5

<210> 125

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 125

Leu Gly Ser Asn Arg Ala Ser

1 5

<210> 126

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 126

Gly Ala Ser Thr Leu Gln Ser

1 5

<210> 127

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 127

Asp Ala Ser Asn Leu Glu Thr

1 5

<210> 128

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 128

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 129

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic peptide"

<400> 129

Met Gln Ala Leu Gln Thr Leu Ile Thr

1 5

<210> 130

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 130

Gln Gln Tyr Glu Asn Leu Pro Leu Thr

1 5

<210> 131

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 131

Gln Gln Ala Ser Ile Phe Pro Pro Thr

1 5

<210> 132

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 132

Met Gln Ala Leu Gln Thr Pro Phe Thr

1 5

<210> 133

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 133

Gln Gln Tyr Asp Asp Leu Pro Leu Thr

1 5

<210> 134

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 134

Met Gln Ala Leu Gln Thr Pro Thr

1 5

<210> 135

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 135

Gln Gln Ser Asn Asn Phe Pro Pro Thr

1 5

<210> 136

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 136

Gln Gln Ser Asp Asp Leu Pro His Thr

1 5

<210> 137

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 137

Gln Gln Ser Tyr Ser Thr Pro Leu Thr

1 5

<210> 138

<211> 9742

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 138

gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 60

gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 120

tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 180

acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 240

aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 300

cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 360

gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 420

cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 480

tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 540

tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 600

ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 660

tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 720

gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 780

ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 840

tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 900

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 960

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 1020

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 1080

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 1140

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 1200

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 1260

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 1320

ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 1380

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 1440

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 1500

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 1560

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 1620

gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 1680

cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 1740

gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 1800

gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 1860

gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca 1920

agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc 1980

ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca 2040

aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt 2100

attaggaagg caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg 2160

cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct ggttagacca 2220

gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 2280

cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag 2340

atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac 2400

ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg 2460

cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga 2520

ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg 2580

cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata 2640

gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca 2700

gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa 2760

cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata 2820

aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc 2880

gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag 2940

aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa 3000

ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct 3060

tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca 3120

ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga 3180

ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat 3240

cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg 3300

aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga 3360

acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag 3420

cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt 3480

attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg 3540

gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc 3600

tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca 3660

cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag 3720

agacagagac agatccattc gattagtgaa cggatctcga cggtatcgat tagactgtag 3780

cccaggaata tggcagctag attgtacaca tttagaagga aaagttatct tggtagcagt 3840

tcatgtagcc agtggatata tagaagcaga agtaattcca gcagagacag ggcaagaaac 3900

agcatacttc ctcttaaaat tagcaggaag atggccagta aaaacagtac atacagacaa 3960

tggcagcaat ttcaccagta ctacagttaa ggccgcctgt tggtgggcgg ggatcaagca 4020

ggaatttggc attccctaca atccccaaag tcaaggagta atagaatcta tgaataaaga 4080

attaaagaaa attataggac aggtaagaga tcaggctgaa catcttaaga cagcagtaca 4140

aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg 4200

ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat 4260

tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggctgca 4320

tacgcgtcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 4380

gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta 4440

aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg 4500

tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca 4560

caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc 4620

gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt 4680

ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga 4740

gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc 4800

tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg 4860

ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg 4920

gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg 4980

cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct 5040

gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc 5100

cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc 5160

tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa 5220

agggcctttc cgtcctcagc cgtcgcttca tgtgactcca ctgagtaccg ggcgccgtcc 5280

aggcacctcg attagttctc gtgcttttgg agtacgtcgt ctttaggttg gggggagggg 5340

ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg 5400

cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc 5460

aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg agctagctct 5520

agagccacca tggccctgcc tgtgacagcc ctgctgctgc ctctggctct gctgctgcat 5580

gccgctagac ccggatccaa catcatgctg acccagagcc ctagcagcct ggccgtgtct 5640

gccggcgaga aagtgaccat gagctgcaag agcagccaga gcgtgttctt cagcagctcc 5700

cagaagaact acctggcctg gtatcagcag atccccggcc agagccccaa gctgctgatc 5760

tactgggcca gcaccagaga aagcggcgtg cccgatagat tcaccggcag cggctctggc 5820

accgacttca ccctgacaat cagcagcgtg cagagcgagg acctggccat ctactactgc 5880

caccagtacc tgagcagccg gacctttggc ggaggcacca agctggaaat caagagaggc 5940

ggcggaggct caggcggagg cggatctagt ggcggaggat ctcaggtgca gctgcagcag 6000

ccaggcgccg aggtcgtgaa acctggcgcc tctgtgaaga tgtcctgcaa ggccagcggc 6060

tacaccttca ccagctacta catccactgg atcaagcaga cccctggaca gggcctggaa 6120

tgggtgggag tgatctaccc cggcaacgac gacatcagct acaaccagaa gttcaagggc 6180

aaggccaccc tgaccgccga caagtctagc accaccgcct acatgcagct gtccagcctg 6240

accagcgagg acagcgccgt gtactactgc gccagagaag tgcggctgcg gtacttcgat 6300

gtgtggggag ccggcaccac cgtgaccgtg tcatcttccg gagagagcaa gtacggccct 6360

ccctgccccc cttgccctgc ccccgagttc ctgggcggac ccagcgtgtt cctgttcccc 6420

cccaagccca aggacaccct gatgatcagc cggacccccg aggtgacctg tgtggtggtg 6480

gacgtgtccc aggaggaccc cgaggtccag ttcaactggt acgtggacgg cgtggaggtg 6540

cacaacgcca agaccaagcc ccgggaggag cagttcaata gcacctaccg ggtggtgtcc 6600

gtgctgaccg tgctgcacca ggactggctg aacggcaagg aatacaagtg taaggtgtcc 6660

aacaagggcc tgcccagcag catcgagaaa accatcagca aggccaaggg ccagcctcgg 6720

gagccccagg tgtacaccct gccccctagc caagaggaga tgaccaagaa ccaggtgtcc 6780

ctgacctgcc tggtgaaggg cttctacccc agcgacatcg ccgtggagtg ggagagcaac 6840

ggccagcccg agaacaacta caagaccacc ccccctgtgc tggacagcga cggcagcttc 6900

ttcctgtaca gccggctgac cgtggacaag agccggtggc aggagggcaa cgtctttagc 6960

tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaagagcct gagcctgtcc 7020

ctgggcaaga tgatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg 7080

tcactggtta tcacccttta ctgcaaacgg ggcagaaaga aactcctgta tatattcaaa 7140

caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 7200

ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag cgcagacgcc 7260

cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 7320

gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 7380

aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 7440

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 7500

cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 7560

cctcgctaag tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt 7620

cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat 7680

gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct 7740

ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct 7800

gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc 7860

gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg 7920

acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc 7980

tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac 8040

gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg 8100

cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc 8160

ccgcctggaa ttcgagctcg gtacctttaa gaccaatgac ttacaaggca gctgtagatc 8220

ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac 8280

aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc 8340

tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc 8400

aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt 8460

agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat 8520

aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg 8580

gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 8640

ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc 8700

gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 8760

ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt 8820

ttttggaggc ctagctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg 8880

ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 8940

tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 9000

tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 9060

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 9120

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 9180

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 9240

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 9300

ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 9360

aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 9420

ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 9480

attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 9540

ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 9600

cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 9660

cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 9720

aaagatgctg aagatcagtt gg 9742

<210> 139

<211> 2037

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 139

atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca tgccgctaga 60

cccggatcca acatcatgct gacccagagc cctagcagcc tggccgtgtc tgccggcgag 120

aaagtgacca tgagctgcaa gagcagccag agcgtgttct tcagcagctc ccagaagaac 180

tacctggcct ggtatcagca gatccccggc cagagcccca agctgctgat ctactgggcc 240

agcaccagag aaagcggcgt gcccgataga ttcaccggca gcggctctgg caccgacttc 300

accctgacaa tcagcagcgt gcagagcgag gacctggcca tctactactg ccaccagtac 360

ctgagcagcc ggacctttgg cggaggcacc aagctggaaa tcaagagagg cggcggaggc 420

tcaggcggag gcggatctag tggcggagga tctcaggtgc agctgcagca gccaggcgcc 480

gaggtcgtga aacctggcgc ctctgtgaag atgtcctgca aggccagcgg ctacaccttc 540

accagctact acatccactg gatcaagcag acccctggac agggcctgga atgggtggga 600

gtgatctacc ccggcaacga cgacatcagc tacaaccaga agttcaaggg caaggccacc 660

ctgaccgccg acaagtctag caccaccgcc tacatgcagc tgtccagcct gaccagcgag 720

gacagcgccg tgtactactg cgccagagaa gtgcggctgc ggtacttcga tgtgtgggga 780

gccggcacca ccgtgaccgt gtcatcttcc ggagagagca agtacggccc tccctgcccc 840

ccttgccctg cccccgagtt cctgggcgga cccagcgtgt tcctgttccc ccccaagccc 900

aaggacaccc tgatgatcag ccggaccccc gaggtgacct gtgtggtggt ggacgtgtcc 960

caggaggacc ccgaggtcca gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc 1020

aagaccaagc cccgggagga gcagttcaat agcacctacc gggtggtgtc cgtgctgacc 1080

gtgctgcacc aggactggct gaacggcaag gaatacaagt gtaaggtgtc caacaagggc 1140

ctgcccagca gcatcgagaa aaccatcagc aaggccaagg gccagcctcg ggagccccag 1200

gtgtacaccc tgccccctag ccaagaggag atgaccaaga accaggtgtc cctgacctgc 1260

ctggtgaagg gcttctaccc cagcgacatc gccgtggagt gggagagcaa cggccagccc 1320

gagaacaact acaagaccac cccccctgtg ctggacagcg acggcagctt cttcctgtac 1380

agccggctga ccgtggacaa gagccggtgg caggagggca acgtctttag ctgctccgtg 1440

atgcacgagg ccctgcacaa ccactacacc cagaagagcc tgagcctgtc cctgggcaag 1500

atgatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 1560

atcacccttt actgcaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 1620

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 1680

gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1740

aagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1800

gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 1860

cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1920

attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1980

agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgc 2037

<210> 140

<211> 679

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 140

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gly Ser Asn Ile Met Leu Thr Gln Ser Pro Ser

20 25 30

Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser

35 40 45

Ser Gln Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu Ala Trp

50 55 60

Tyr Gln Gln Ile Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala

65 70 75 80

Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

85 90 95

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ser Glu Asp Leu

100 105 110

Ala Ile Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr Phe Gly Gly

115 120 125

Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Ser Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala

145 150 155 160

Glu Val Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser

165 170 175

Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro

180 185 190

Gly Gln Gly Leu Glu Trp Val Gly Val Ile Tyr Pro Gly Asn Asp Asp

195 200 205

Ile Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp

210 215 220

Lys Ser Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu

225 230 235 240

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe

245 250 255

Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ser Gly Glu

260 265 270

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

275 280 285

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

290 295 300

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

305 310 315 320

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

325 330 335

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

340 345 350

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

355 360 365

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

370 375 380

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

385 390 395 400

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

405 410 415

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

420 425 430

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

435 440 445

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

450 455 460

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

465 470 475 480

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

485 490 495

Ser Leu Gly Lys Met Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

500 505 510

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

515 520 525

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

530 535 540

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

545 550 555 560

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

565 570 575

Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

580 585 590

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

595 600 605

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

610 615 620

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

625 630 635 640

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

645 650 655

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

660 665 670

Met Gln Ala Leu Pro Pro Arg

675

<210> 141

<211> 807

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 141

atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca tgccgctaga 60

cccggatcca acatcatgct gacccagagc cctagcagcc tggccgtgtc tgccggcgag 120

aaagtgacca tgagctgcaa gagcagccag agcgtgttct tcagcagctc ccagaagaac 180

tacctggcct ggtatcagca gatccccggc cagagcccca agctgctgat ctactgggcc 240

agcaccagag aaagcggcgt gcccgataga ttcaccggca gcggctctgg caccgacttc 300

accctgacaa tcagcagcgt gcagagcgag gacctggcca tctactactg ccaccagtac 360

ctgagcagcc ggacctttgg cggaggcacc aagctggaaa tcaagagagg cggcggaggc 420

tcaggcggag gcggatctag tggcggagga tctcaggtgc agctgcagca gccaggcgcc 480

gaggtcgtga aacctggcgc ctctgtgaag atgtcctgca aggccagcgg ctacaccttc 540

accagctact acatccactg gatcaagcag acccctggac agggcctgga atgggtggga 600

gtgatctacc ccggcaacga cgacatcagc tacaaccaga agttcaaggg caaggccacc 660

ctgaccgccg acaagtctag caccaccgcc tacatgcagc tgtccagcct gaccagcgag 720

gacagcgccg tgtactactg cgccagagaa gtgcggctgc ggtacttcga tgtgtgggga 780

gccggcacca ccgtgaccgt gtcatct 807

<210> 142

<211> 269

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 142

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gly Ser Asn Ile Met Leu Thr Gln Ser Pro Ser

20 25 30

Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser

35 40 45

Ser Gln Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu Ala Trp

50 55 60

Tyr Gln Gln Ile Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala

65 70 75 80

Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

85 90 95

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ser Glu Asp Leu

100 105 110

Ala Ile Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr Phe Gly Gly

115 120 125

Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Ser Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala

145 150 155 160

Glu Val Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser

165 170 175

Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro

180 185 190

Gly Gln Gly Leu Glu Trp Val Gly Val Ile Tyr Pro Gly Asn Asp Asp

195 200 205

Ile Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp

210 215 220

Lys Ser Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu

225 230 235 240

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe

245 250 255

Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser

260 265

<210> 143

<211> 9742

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 143

gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 60

gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 120

tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 180

acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 240

aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 300

cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 360

gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 420

cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 480

tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 540

tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 600

ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 660

tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 720

gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 780

ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 840

tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 900

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 960

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 1020

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 1080

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 1140

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 1200

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 1260

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 1320

ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 1380

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 1440

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 1500

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 1560

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 1620

gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 1680

cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 1740

gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 1800

gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 1860

gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca 1920

agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc 1980

ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca 2040

aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt 2100

attaggaagg caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg 2160

cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct ggttagacca 2220

gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 2280

cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag 2340

atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac 2400

ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg 2460

cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga 2520

ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg 2580

cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata 2640

gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca 2700

gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa 2760

cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata 2820

aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc 2880

gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag 2940

aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa 3000

ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct 3060

tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca 3120

ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga 3180

ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat 3240

cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg 3300

aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga 3360

acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag 3420

cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt 3480

attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg 3540

gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc 3600

tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca 3660

cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag 3720

agacagagac agatccattc gattagtgaa cggatctcga cggtatcgat tagactgtag 3780

cccaggaata tggcagctag attgtacaca tttagaagga aaagttatct tggtagcagt 3840

tcatgtagcc agtggatata tagaagcaga agtaattcca gcagagacag ggcaagaaac 3900

agcatacttc ctcttaaaat tagcaggaag atggccagta aaaacagtac atacagacaa 3960

tggcagcaat ttcaccagta ctacagttaa ggccgcctgt tggtgggcgg ggatcaagca 4020

ggaatttggc attccctaca atccccaaag tcaaggagta atagaatcta tgaataaaga 4080

attaaagaaa attataggac aggtaagaga tcaggctgaa catcttaaga cagcagtaca 4140

aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg 4200

ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat 4260

tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggctgca 4320

tacgcgtcgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 4380

gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta 4440

aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg 4500

tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca 4560

caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc 4620

gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt 4680

ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga 4740

gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc 4800

tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg 4860

ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg 4920

gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg 4980

cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct 5040

gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc 5100

cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc 5160

tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa 5220

agggcctttc cgtcctcagc cgtcgcttca tgtgactcca ctgagtaccg ggcgccgtcc 5280

aggcacctcg attagttctc gtgcttttgg agtacgtcgt ctttaggttg gggggagggg 5340

ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg 5400

cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc 5460

aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg agctagctct 5520

agagccacca tggccctgcc tgtgacagcc ctgctgctgc ctctggctct gctgctgcat 5580

gccgctagac ccggatccga gatcgtgctg acacagagcc ctggaagcct ggccgtgtct 5640

cctggcgagc gcgtgacaat gagctgcaag agcagccaga gcgtgttctt cagcagctcc 5700

cagaagaact acctggcctg gtatcagcag atccccggcc agagccccag actgctgatc 5760

tactgggcca gcaccagaga aagcggcgtg cccgatagat tcaccggcag cggctctggc 5820

accgacttca ccctgacaat cagcagcgtg cagcccgagg acctggccat ctactactgc 5880

caccagtacc tgagcagccg gacctttggc cagggcacca agctggaaat caagagaggc 5940

ggcggaggct ctggcggagg cggatctagt ggcggaggat ctcaggtgca gctgcagcag 6000

cctggcgccg aggtcgtgaa acctggcgcc tctgtgaaga tgtcctgcaa ggccagcggc 6060

tacaccttca ccagctacta catccactgg atcaagcaga cccctggaca gggcctggaa 6120

tgggtgggag tgatctaccc cggcaacgac gacatcagct acaaccagaa gttccagggc 6180

aaggccaccc tgaccgccga caagtctagc accaccgcct acatgcagct gtccagcctg 6240

accagcgagg acagcgccgt gtactactgc gccagagaag tgcggctgcg gtacttcgat 6300

gtgtggggcc agggaaccac cgtgaccgtg tcatcttccg gagagagcaa gtacggccct 6360

ccctgccccc cttgccctgc ccccgagttc ctgggcggac ccagcgtgtt cctgttcccc 6420

cccaagccca aggacaccct gatgatcagc cggacccccg aggtgacctg tgtggtggtg 6480

gacgtgtccc aggaggaccc cgaggtccag ttcaactggt acgtggacgg cgtggaggtg 6540

cacaacgcca agaccaagcc ccgggaggag cagttcaata gcacctaccg ggtggtgtcc 6600

gtgctgaccg tgctgcacca ggactggctg aacggcaagg aatacaagtg taaggtgtcc 6660

aacaagggcc tgcccagcag catcgagaaa accatcagca aggccaaggg ccagcctcgg 6720

gagccccagg tgtacaccct gccccctagc caagaggaga tgaccaagaa ccaggtgtcc 6780

ctgacctgcc tggtgaaggg cttctacccc agcgacatcg ccgtggagtg ggagagcaac 6840

ggccagcccg agaacaacta caagaccacc ccccctgtgc tggacagcga cggcagcttc 6900

ttcctgtaca gccggctgac cgtggacaag agccggtggc aggagggcaa cgtctttagc 6960

tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaagagcct gagcctgtcc 7020

ctgggcaaga tgatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg 7080

tcactggtta tcacccttta ctgcaaacgg ggcagaaaga aactcctgta tatattcaaa 7140

caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 7200

ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag cgcagacgcc 7260

cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 7320

gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 7380

aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 7440

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 7500

cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 7560

cctcgctaag tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt 7620

cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat 7680

gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct 7740

ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct 7800

gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc 7860

gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg 7920

acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc 7980

tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac 8040

gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg 8100

cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc 8160

ccgcctggaa ttcgagctcg gtacctttaa gaccaatgac ttacaaggca gctgtagatc 8220

ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac 8280

aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc 8340

tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc 8400

aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt 8460

agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat 8520

aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg 8580

gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 8640

ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc 8700

gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 8760

ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt 8820

ttttggaggc ctagctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg 8880

ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 8940

tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 9000

tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 9060

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 9120

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 9180

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 9240

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 9300

ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 9360

aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 9420

ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 9480

attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 9540

ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 9600

cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 9660

cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 9720

aaagatgctg aagatcagtt gg 9742

<210> 144

<211> 2037

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 144

atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca tgccgctaga 60

cccggatccg agatcgtgct gacacagagc cctggaagcc tggccgtgtc tcctggcgag 120

cgcgtgacaa tgagctgcaa gagcagccag agcgtgttct tcagcagctc ccagaagaac 180

tacctggcct ggtatcagca gatccccggc cagagcccca gactgctgat ctactgggcc 240

agcaccagag aaagcggcgt gcccgataga ttcaccggca gcggctctgg caccgacttc 300

accctgacaa tcagcagcgt gcagcccgag gacctggcca tctactactg ccaccagtac 360

ctgagcagcc ggacctttgg ccagggcacc aagctggaaa tcaagagagg cggcggaggc 420

tctggcggag gcggatctag tggcggagga tctcaggtgc agctgcagca gcctggcgcc 480

gaggtcgtga aacctggcgc ctctgtgaag atgtcctgca aggccagcgg ctacaccttc 540

accagctact acatccactg gatcaagcag acccctggac agggcctgga atgggtggga 600

gtgatctacc ccggcaacga cgacatcagc tacaaccaga agttccaggg caaggccacc 660

ctgaccgccg acaagtctag caccaccgcc tacatgcagc tgtccagcct gaccagcgag 720

gacagcgccg tgtactactg cgccagagaa gtgcggctgc ggtacttcga tgtgtggggc 780

cagggaacca ccgtgaccgt gtcatcttcc ggagagagca agtacggccc tccctgcccc 840

ccttgccctg cccccgagtt cctgggcgga cccagcgtgt tcctgttccc ccccaagccc 900

aaggacaccc tgatgatcag ccggaccccc gaggtgacct gtgtggtggt ggacgtgtcc 960

caggaggacc ccgaggtcca gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc 1020

aagaccaagc cccgggagga gcagttcaat agcacctacc gggtggtgtc cgtgctgacc 1080

gtgctgcacc aggactggct gaacggcaag gaatacaagt gtaaggtgtc caacaagggc 1140

ctgcccagca gcatcgagaa aaccatcagc aaggccaagg gccagcctcg ggagccccag 1200

gtgtacaccc tgccccctag ccaagaggag atgaccaaga accaggtgtc cctgacctgc 1260

ctggtgaagg gcttctaccc cagcgacatc gccgtggagt gggagagcaa cggccagccc 1320

gagaacaact acaagaccac cccccctgtg ctggacagcg acggcagctt cttcctgtac 1380

agccggctga ccgtggacaa gagccggtgg caggagggca acgtctttag ctgctccgtg 1440

atgcacgagg ccctgcacaa ccactacacc cagaagagcc tgagcctgtc cctgggcaag 1500

atgatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 1560

atcacccttt actgcaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 1620

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 1680

gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1740

aagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1800

gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 1860

cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1920

attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1980

agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgc 2037

<210> 145

<211> 679

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 145

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly

20 25 30

Ser Leu Ala Val Ser Pro Gly Glu Arg Val Thr Met Ser Cys Lys Ser

35 40 45

Ser Gln Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu Ala Trp

50 55 60

Tyr Gln Gln Ile Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Trp Ala

65 70 75 80

Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

85 90 95

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Leu

100 105 110

Ala Ile Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr Phe Gly Gln

115 120 125

Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Ser Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala

145 150 155 160

Glu Val Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser

165 170 175

Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro

180 185 190

Gly Gln Gly Leu Glu Trp Val Gly Val Ile Tyr Pro Gly Asn Asp Asp

195 200 205

Ile Ser Tyr Asn Gln Lys Phe Gln Gly Lys Ala Thr Leu Thr Ala Asp

210 215 220

Lys Ser Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu

225 230 235 240

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe

245 250 255

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ser Gly Glu

260 265 270

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

275 280 285

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

290 295 300

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

305 310 315 320

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

325 330 335

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

340 345 350

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

355 360 365

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

370 375 380

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

385 390 395 400

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

405 410 415

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

420 425 430

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

435 440 445

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

450 455 460

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

465 470 475 480

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

485 490 495

Ser Leu Gly Lys Met Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

500 505 510

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

515 520 525

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

530 535 540

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

545 550 555 560

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

565 570 575

Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

580 585 590

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

595 600 605

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

610 615 620

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

625 630 635 640

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

645 650 655

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

660 665 670

Met Gln Ala Leu Pro Pro Arg

675

<210> 146

<211> 738

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 146

gagatcgtgc tgacacagag ccctggaagc ctggccgtgt ctcctggcga gcgcgtgaca 60

atgagctgca agagcagcca gagcgtgttc ttcagcagct cccagaagaa ctacctggcc 120

tggtatcagc agatccccgg ccagagcccc agactgctga tctactgggc cagcaccaga 180

gaaagcggcg tgcccgatag attcaccggc agcggctctg gcaccgactt caccctgaca 240

atcagcagcg tgcagcccga ggacctggcc atctactact gccaccagta cctgagcagc 300

cggacctttg gccagggcac caagctggaa atcaagagag gcggcggagg ctctggcgga 360

ggcggatcta gtggcggagg atctcaggtg cagctgcagc agcctggcgc cgaggtcgtg 420

aaacctggcg cctctgtgaa gatgtcctgc aaggccagcg gctacacctt caccagctac 480

tacatccact ggatcaagca gacccctgga cagggcctgg aatgggtggg agtgatctac 540

cccggcaacg acgacatcag ctacaaccag aagttccagg gcaaggccac cctgaccgcc 600

gacaagtcta gcaccaccgc ctacatgcag ctgtccagcc tgaccagcga ggacagcgcc 660

gtgtactact gcgccagaga agtgcggctg cggtacttcg atgtgtgggg ccagggaacc 720

accgtgaccg tgtcatct 738

<210> 147

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 147

Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser

20 25 30

Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln

35 40 45

Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln

85 90 95

Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser

115 120 125

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala

130 135 140

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

145 150 155 160

Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val

165 170 175

Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe

180 185 190

Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr

195 200 205

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

210 215 220

Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr

225 230 235 240

Thr Val Thr Val Ser Ser

245

<210> 148

<211> 132

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 148

Asp Val Pro Asp Tyr Ala Ser Leu Gly Gly Pro Ser Ser Pro Lys Lys

1 5 10 15

Lys Arg Lys Val Ser Arg Gly Val Gln Val Glu Thr Ile Ser Pro Gly

20 25 30

Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr

35 40 45

Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg

50 55 60

Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly

65 70 75 80

Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu

85 90 95

Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile

100 105 110

Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu

115 120 125

Glu Thr Ser Tyr

130

<210> 149

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 149

Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys

1 5 10 15

Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly

20 25 30

Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met

35 40 45

Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln

50 55 60

Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala

65 70 75 80

Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu

85 90 95

Val Phe Asp Val Glu Leu Leu Lys Leu Glu Thr Ser

100 105

<210> 150

<211> 93

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 150

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys

85 90

<210> 151

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 151

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Ile Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 152

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 152

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Leu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 153

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 153

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 154

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<220>

<221> MOD_RES

<222> (12)..(12)

<223> any amino acid

<220>

<221> MOD_RES

<222> (78)..(78)

<223> any amino acid

<400> 154

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Xaa Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Xaa Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 155

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 155

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Ile Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 156

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 156

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Leu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 157

<211> 1132

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 157

Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser

1 5 10 15

His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly

20 25 30

Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg

35 40 45

Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro

50 55 60

Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu

65 70 75 80

Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val

85 90 95

Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro

100 105 110

Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr

115 120 125

Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val

130 135 140

Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val

145 150 155 160

Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr

165 170 175

Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly

180 185 190

Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg

195 200 205

Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg

210 215 220

Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg

225 230 235 240

Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp

245 250 255

Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val

260 265 270

Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala

275 280 285

Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His

290 295 300

Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro

305 310 315 320

Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly

325 330 335

Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro

340 345 350

Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser

355 360 365

Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln

370 375 380

Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His

385 390 395 400

Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg

405 410 415

Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln

420 425 430

Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu

435 440 445

Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe

450 455 460

Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser

465 470 475 480

Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser

485 490 495

Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met

500 505 510

Ser Val Arg Gly Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys

515 520 525

Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe

530 535 540

Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe

545 550 555 560

Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr

565 570 575

Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His

580 585 590

Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln

595 600 605

His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile

610 615 620

Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val

625 630 635 640

Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser

645 650 655

Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg

660 665 670

Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg

675 680 685

Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro

690 695 700

Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile

705 710 715 720

Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln

725 730 735

Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His

740 745 750

Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp

755 760 765

Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser

770 775 780

Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu

785 790 795 800

Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His

805 810 815

Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro

820 825 830

Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp

835 840 845

Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu

850 855 860

Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala

865 870 875 880

Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys

885 890 895

Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu

900 905 910

Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe

915 920 925

Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser

930 935 940

Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe

945 950 955 960

Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly

965 970 975

Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn

980 985 990

Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln

995 1000 1005

Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln

1010 1015 1020

Gln Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp

1025 1030 1035

Thr Ala Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly

1040 1045 1050

Met Ser Leu Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu

1055 1060 1065

Ala Val Gln Trp Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr

1070 1075 1080

Arg His Arg Val Thr Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr

1085 1090 1095

Ala Gln Thr Gln Leu Ser Arg Lys Leu Pro Gly Thr Thr Leu Thr

1100 1105 1110

Ala Leu Glu Ala Ala Ala Asn Pro Ala Leu Pro Ser Asp Phe Lys

1115 1120 1125

Thr Ile Leu Asp

1130

<210> 158

<211> 4027

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 158

caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcgatgc 60

cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactac cgcgaggtgc 120

tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg gtgcagcgcg 180

gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg ccctgggacg 240

cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag gagctggtgg 300

cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc ttcggcttcg 360

cgctgctgga cggggcccgc gggggccccc ccgaggcctt caccaccagc gtgcgcagct 420

acctgcccaa cacggtgacc gacgcactgc gggggagcgg ggcgtggggg ctgctgttgc 480

gccgcgtggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc tttgtgctgg 540

tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca 600

ctcaggcccg gcccccgcca cacgctagtg gaccccgaag gcgtctggga tgcgaacggg 660

cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagcc ccgggtgcga 720

ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggccc aggcgtggcg 780

ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccac ccgggcagga 840

cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacc tgccagaccc gccgaagaag 900

ccacctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc 960

agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac acgccttgtc 1020

ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaag gagcagctgc 1080

ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcgg aggctcgtgg 1140

agaccatctt tctgggttcc aggccctgga tgccagggac tccccgcagg ttgccccgcc 1200

tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc 1260

agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg gtcaccccag 1320

cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggccccc gaggaggagg 1380

acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcagcccc tggcaggtgt 1440

acggcttcgt gcgggcctgc ctgcgccggc tggtgccccc aggcctctgg ggctccaggc 1500

acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctgggg aagcatgcca 1560

agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct tggctgcgca 1620

ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag gagatcctgg 1680

ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcagg tctttctttt 1740

atgtcacgga gaccacgttt caaaagaaca ggctcttttt ctaccggaag agtgtctgga 1800

gcaagttgca aagcattgga atcagacagc acttgaagag ggtgcagctg cgggagctgt 1860

cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacg tccagactcc 1920

gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactac gtcgtgggag 1980

ccagaacgtt ccgcagagaa aagagggccg agcgtctcac ctcgagggtg aaggcactgt 2040

tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc tctgtgctgg 2100

gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg gcccaggacc 2160

cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac accatccccc 2220

aggacaggct cacggaggtc atcgccagca tcatcaaacc ccagaacacg tactgcgtgc 2280

gtcggtatgc cgtggtccag aaggccgccc atgggcacgt ccgcaaggcc ttcaagagcc 2340

acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct cacctgcagg 2400

agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg aatgaggcca 2460

gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg cgcatcaggg 2520

gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctcc acgctgctct 2580

gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcgg cgggacgggc 2640

tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa 2700

ccttcctcag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga 2760

agacagtggt gaacttccct gtagaagacg aggccctggg tggcacggct tttgttcaga 2820

tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccgg accctggagg 2880

tgcagagcga ctactccagc tatgcccgga cctccatcag agccagtctc accttcaacc 2940

gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tggggtcttg cggctgaagt 3000

gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgc accaacatct 3060

acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag ctcccatttc 3120

atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgac acggcctccc 3180

tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggcc aagggcgccg 3240

ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattc ctgctcaagc 3300

tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggaca gcccagacgc 3360

agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca gccaacccgg 3420

cactgccctc agacttcaag accatcctgg actgatggcc acccgcccac agccaggccg 3480

agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagg gaggggcggc 3540

ccacacccag gcccgcaccg ctgggagtct gaggcctgag tgagtgtttg gccgaggcct 3600

gcatgtccgg ctgaaggctg agtgtccggc tgaggcctga gcgagtgtcc agccaagggc 3660

tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcg gctccacccc 3720

agggccagct tttcctcacc aggagcccgg cttccactcc ccacatagga atagtccatc 3780

cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccac ccccaccatc 3840

caggtggaga ccctgagaag gaccctggga gctctgggaa tttggagtga ccaaaggtgt 3900

gccctgtaca caggcgagga ccctgcacct ggatgggggt ccctgtgggt caaattgggg 3960

ggaggtgctg tgggagtaaa atactgaata tatgagtttt tcagttttga aaaaaaaaaa 4020

aaaaaaa 4027

<210> 159

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 159

ggaggtccct caccttcta 19

<210> 160

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 160

cggaggatct tatgctgaa 19

<210> 161

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 161

cccgcttcca gatcataca 19

<210> 162

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 162

ggagacctca acaagatat 19

<210> 163

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 163

aaggcatggt cattggtat 19

<210> 164

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 164

gcatggtcat tggtatcat 19

<210> 165

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 165

ggtcattggt atcatgagt 19

<210> 166

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 166

cctagtgggt atccctgta 19

<210> 167

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 167

gaggatggac attgttctt 19

<210> 168

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 168

gcatgcaggc tacagttca 19

<210> 169

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 169

ccagcacatg cactgttga 19

<210> 170

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 170

cacatgcact gttgagtga 19

<210> 171

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 171

ctggaggtcc ctcaccttct a 21

<210> 172

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 172

gtcggaggat cttatgctga a 21

<210> 173

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 173

tgcccgcttc cagatcatac a 21

<210> 174

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 174

ctggagacct caacaagata t 21

<210> 175

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 175

tcaaggcatg gtcattggta t 21

<210> 176

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 176

aggcatggtc attggtatca t 21

<210> 177

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 177

atggtcattg gtatcatgag t 21

<210> 178

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 178

gccctagtgg gtatccctgt a 21

<210> 179

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 179

atgaggatgg acattgttct t 21

<210> 180

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 180

gagcatgcag gctacagttc a 21

<210> 181

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 181

ttccagcaca tgcactgttg a 21

<210> 182

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 182

agcacatgca ctgttgagtg a 21

<210> 183

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 183

tagaaggtga gggacctcca g 21

<210> 184

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 184

ttcagcataa gatcctccga c 21

<210> 185

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 185

tgtatgatct ggaagcgggc a 21

<210> 186

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 186

atatcttgtt gaggtctcca g 21

<210> 187

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 187

ataccaatga ccatgccttg a 21

<210> 188

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 188

atgataccaa tgaccatgcc t 21

<210> 189

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 189

atggtcattg gtatcatgag t 21

<210> 190

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 190

gccctagtgg gtatccctgt a 21

<210> 191

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 191

atgaggatgg acattgttct t 21

<210> 192

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 192

gagcatgcag gctacagttc a 21

<210> 193

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 193

ttccagcaca tgcactgttg a 21

<210> 194

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 194

agcacatgca ctgttgagtg a 21

<210> 195

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 195

tagaaggtga gggacctcc 19

<210> 196

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 196

ttcagcataa gatcctccg 19

<210> 197

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 197

tgtatgatct ggaagcggg 19

<210> 198

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 198

atatcttgtt gaggtctcc 19

<210> 199

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 199

ataccaatga ccatgcctt 19

<210> 200

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 200

atgataccaa tgaccatgc 19

<210> 201

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 201

atggtcattg gtatcatga 19

<210> 202

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 202

gccctagtgg gtatccctg 19

<210> 203

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 203

atgaggatgg acattgttc 19

<210> 204

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 204

gagcatgcag gctacagtt 19

<210> 205

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 205

ttccagcaca tgcactgtt 19

<210> 206

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 206

agcacatgca ctgttgagt 19

<210> 207

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 207

ggccaggatg gttcttaga 19

<210> 208

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 208

gcttcgtgct aaactggta 19

<210> 209

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 209

gggcgtgact tccacatga 19

<210> 210

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 210

caggcctaga gaagtttca 19

<210> 211

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 211

cttggaaccc attcctgaa 19

<210> 212

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 212

ggaacccatt cctgaaatt 19

<210> 213

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 213

gaacccattc ctgaaatta 19

<210> 214

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 214

aacccattcc tgaaattat 19

<210> 215

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 215

acccattcct gaaattatt 19

<210> 216

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 216

cccattcctg aaattattt 19

<210> 217

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 217

ctgtggttct attatatta 19

<210> 218

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 218

aaatatgaga gcatgctaa 19

<210> 219

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 219

tctaagaacc atcctggcc 19

<210> 220

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 220

taccagttta gcacgaagc 19

<210> 221

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 221

tcatgtggaa gtcacgccc 19

<210> 222

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 222

tgaaacttct ctaggcctg 19

<210> 223

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 223

ttcaggaatg ggttccaag 19

<210> 224

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 224

aatttcagga atgggttcc 19

<210> 225

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 225

taatttcagg aatgggttc 19

<210> 226

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 226

ataatttcag gaatgggtt 19

<210> 227

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 227

aataatttca ggaatgggt 19

<210> 228

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 228

aaataatttc aggaatggg 19

<210> 229

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 229

taatataata gaaccacag 19

<210> 230

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 230

ttagcatgct ctcatattt 19

<210> 231

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 231

gcggccagga tggttcttag a 21

<210> 232

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 232

gagcttcgtg ctaaactggt a 21

<210> 233

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 233

acgggcgtga cttccacatg a 21

<210> 234

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 234

tgcaggccta gagaagtttc a 21

<210> 235

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 235

tccttggaac ccattcctga a 21

<210> 236

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 236

ttggaaccca ttcctgaaat t 21

<210> 237

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 237

tggaacccat tcctgaaatt a 21

<210> 238

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 238

ggaacccatt cctgaaatta t 21

<210> 239

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 239

gaacccattc ctgaaattat t 21

<210> 240

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 240

aacccattcc tgaaattatt t 21

<210> 241

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 241

ccctgtggtt ctattatatt a 21

<210> 242

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 242

ttaaatatga gagcatgcta a 21

<210> 243

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 243

tctaagaacc atcctggccg c 21

<210> 244

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 244

taccagttta gcacgaagct c 21

<210> 245

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 245

tcatgtggaa gtcacgcccg t 21

<210> 246

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 246

tgaaacttct ctaggcctgc a 21

<210> 247

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 247

ttcaggaatg ggttccaagg a 21

<210> 248

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 248

aatttcagga atgggttcca a 21

<210> 249

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 249

taatttcagg aatgggttcc a 21

<210> 250

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 250

ataatttcag gaatgggttc c 21

<210> 251

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 251

aataatttca ggaatgggtt c 21

<210> 252

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 252

aaataatttc aggaatgggt t 21

<210> 253

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 253

taatataata gaaccacagg g 21

<210> 254

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Oligonucleotides "

<400> 254

ttagcatgct ctcatattta a 21

<210> 255

<211> 750

<212> DNA

<213> human

<400> 255

caagtgcaac tcgtccagtc cggtgcagaa gtcaagaagc caggagaatc actcaagatt 60

agctgcaaag gcagcggcta ctccttcact tcctactgga tcggctgggt gcgccagatg 120

cccggaaagg gactggagtg gatgggaatc atctaccctg gcgatagcga caccagatac 180

tccccgagct ttcaaggcca agtgaccatt tcggccgaca agtcgatctc caccgcgtat 240

ctgcagtgga gctcactgaa ggcttcggac accgccatgt actactgtgc ccggctgggg 300

ggaagcctgc ccgattacgg aatggacgtg tggggccagg gaaccatggt cactgtgtcc 360

tccgcctccg ggggtggagg ctccggtgga ggggggtccg gtggtggagg atcagaaatt 420

gtgctgaccc agtctccgct gtccttgcct gtgaccccgg gcgaacccgc aagcatctcc 480

tgccggtcgt cgcagtccct gcttcactcc aacggctaca actacctcga ttggtacctc 540

cagaagcctg gacagagccc acagctgttg atctacctgg gctcgaaccg ggcctcagga 600

gtgccggaca ggttctccgg ctccgggtcg ggaaccgact tcacgctgaa gatctcccgc 660

gtggaggccg aggacgtggg cgtgtactat tgcatgcagg cgctgcagac ccttattaca 720

ttcggacagg ggactaaggt cgatatcaag 750

<210> 256

<211> 753

<212> DNA

<213> human

<400> 256

caagtccaac tcgtccaatc aggagctgaa gtcaagaagc ctggagcatc cgtgagagtg 60

tcctgtaaag cctccggcta catcttcacc aactactacg tgcactgggt cagacaggcc 120

ccgggccagg gactggaatg gatgggaatc atttccccgt ccggcggatc gcctacttac 180

gcgcaacggc tgcagggccg cgtgaccatg actcgggatc tctccacttc aaccgtgtac 240

atggaactgt ccagccttac atcggaggat actgccgtgt acttctgcgc gagggagtcc 300

cggctgaggg gcaaccgcct cgggctgcag tcaagcatct tcgatcactg gggccagggc 360

accctcgtga ccgtgtccag cgcctcgggg ggaggaggct ccgggggcgg aggttcgggc 420

ggtggtggat ctgacattcg catgactcag tccccacctt cactgtccgc tagcgtgggg 480

gaccgcgtga cgattccgtg ccaagccagc caggacatca acaaccatct gaactggtat 540

cagcagaagc ccggaaaggc cccgcagctg ctgatctacg acacctcgaa tctggagatc 600

ggcgtgccat cccggttctc cggttcggga agcggaaccg actttaccct gactatctcc 660

tccttgcaac ccgaggacat tgccacctac tactgccagc agtacgaaaa ccttcccctg 720

accttcgggg gtggaaccaa agtggagatc aag 753

<210> 257

<211> 750

<212> DNA

<213> human

<400> 257

caagtgcagc tcgtccaatc cggtgcagaa gtgaagaagc ctggcgaatc cctgaagatc 60

tcatgcaaag gctcgggata cagcttcacc tcatattgga ttggatgggt cagacagatg 120

ccaggaaagg gtctggagtg gatgggaatc atctacccgg gagacagcga tacccggtac 180

tccccgagct tccagggaca ggtcaccatc tcggccgaca agtccattac tactgcctac 240

ttgcaatggt cctcgctgcg cgcctccgat agcgccatgt actactgcgc gagaggcggc 300

tactccgact acgactacta cttcgatttc tggggacagg ggacactcgt gactgtgtcc 360

tccgcgtcgg gtggcggcgg ctcgggtgga ggaggaagcg gagggggagg ctccgaaatt 420

gtgatgaccc agtcacccct gtcgctccct gtgactcctg gggaaccggc ctccatctcc 480

tgccggagct cacagagcct gctgcactcc aacggataca actacctcga ttggtacctt 540

cagaagcccg gccagtcgcc ccagctgctg atctacctgg ggtccaaccg ggctagcggc 600

gtgccggacc gcttctccgg ttccgggtct ggaaccgact tcacgctgaa aatctccagg 660

gtggaggccg aggacgtggg agtgtattac tgtatgcagg ccctgcaaac ccccttcacc 720

tttggcgggg gcaccaaggt cgagattaag 750

<210> 258

<211> 720

<212> DNA

<213> human

<400> 258

caagtgcaac tcgtccaaag cggtggagat ctcgcccagc ccggaagatc ccttagactc 60

tcatgtgccg ccagcgggtt caccttcgac gactacgcta tgcattgggt gcgccaggcc 120

ccggggaagg gactggaatg ggtggccgtg atttggccgg acggcggaca gaagtactac 180

ggagacagcg tgaaagggcg gttcaccgtg tcgagggaca acccgaagaa taccctctac 240

cttcaaatga actccctgcg cgccgaggac accgcgatct actactgcgt gcgccacttt 300

aacgcatggg attactgggg acaggggact ctggtcactg tgtcctccgc ctctggcggc 360

ggaggttccg gcggtggtgg ctccggtgga ggaggatcgg acatccagct gacccagtcc 420

ccttcctcac tgtcggcgta cgtgggaggc cgggtcacta tcacgtgcca ggcatcccag 480

ggcatttccc agttcctgaa ctggttccag cagaagcccg gaaaggcccc taagctgttg 540

atttccgatg ctagcaacct ggaacccggc gtgccgtcac ggttcagcgg ctccgggtcg 600

ggcaccgact tcaccttcac catcactaac ctccaaccgg aggacatcgc cacctattac 660

tgccagcagt acgatgatct gccactgact ttcggcggcg gaaccaaggt cgaaatcaag 720

<210> 259

<211> 744

<212> DNA

<213> human

<400> 259

caagtgcaac tcgtccaatc cggtggtggt gtcgtgcaac caggaaagtc tcttcgcctc 60

tcatgcgctg ccagcggatt cacgttttcc atcttcgcta tgcactgggt gcggcaggcc 120

ccgggaaagg gactggaatg ggtggcaacc atttcatacg atggatcaaa cgcgttctac 180

gccgactccg tggaaggaag gttcaccatc tcgagagaca actccaagga ctcgctgtat 240

ctgcaaatgg actccctgcg ccctgaggat accgccgtct actactgcgt gaaggccggc 300

gacgggggat acgacgtgtt cgattcgtgg ggccagggaa ctctggtcac cgtgtccagc 360

gcgagcgggg gaggcggatc gggtggtgga gggtccgggg gaggaggctc cgagatcgtg 420

atgactcagt cgccgctctc cctccccgtg acccccggag agccagctag catttcatgt 480

cggagctccc agtccctgct gcactccaac ggctacaatt acctggattg gtacttgcag 540

aagcctgggc agagccctca gctgctgatc tacctcggct cgaacagagc ctccggcgtg 600

ccggaccggt tttccgggag cggcagcggc accgatttca ccttgaaaat ctcccgcgtg 660

gaagccgagg acgtgggcgt gtactattgc atgcaggccc tgcagactcc caccttcggc 720

ccgggaacta aggtcgacat caag 744

<210> 260

<211> 735

<212> DNA

<213> human

<400> 260

gaagtgcaat tggtggaatc tggaggagga ttggtgcaac ctggaggatc tctgagactg 60

tcatgtgccg ccagcggctt cacattttcc tcctacgcga tgtcatgggt ccgccaggca 120

ccggggaaag gactggaatg ggtgtccgcc atttcgggat cgggaggctc gacctactac 180

gccgacagcg tgaagggaag attcactatc tcccgggata actccaagaa tactctgtat 240

ctccaaatga actccctgag ggccgaggat actgccgtgt actactgcgc taaggaaacc 300

gactactacg gctcaggaac cttcgactac tggggccagg gcaccctcgt gaccgtgtcc 360

tcggcctccg gcggcggagg ttcggggggg ggcggttccg ggggaggggg cagcgacatc 420

cagatgaccc agtccccaag ctccctttcc gcgtccgtgg gagatcgcgt gaccatttcg 480

tgccgggcta gccagggcat cggtatctat cttgcgtggt accagcagcg gagcggaaag 540

ccgccccagc tgctgatcca cggcgcctca actctgcaat ccggggtccc cagccggttc 600

agcggtagcg ggtcgggtac cgactttacc ctgactatct cctccctcca accggaggac 660

ttcgcctcct actggtgcca gcagtccaac aacttccctc ccaccttcgg ccagggaacg 720

aaggtcgaga ttaag 735

<210> 261

<211> 732

<212> DNA

<213> human

<400> 261

caagtgcaac tcgtccagtc cggtgcagaa gtgaaaaagc caggagaaag cctcaagatc 60

agctgcaagg gatctgggta cagcttcacc aactactgga tcggctgggt gcgccagatg 120

cccggaaagg gactggagtg gatgggcatt atctaccctg gggactccga cacccggtat 180

tccccgagct tccaaggaca ggtcaccatc tccgccgata agtcgattag cactgcgtac 240

ttgcagtggt caagcctgaa ggcctcggac accgccatgt actactgcgc gagacacggg 300

ccctcgtcct ggggcgaatt tgactactgg ggccagggaa cgcttgtgac cgtgtcgtcc 360

gcgtccgggg gtggaggatc aggaggagga ggctccggtg gtggcggtag cgacatccgg 420

ctgactcagt ccccttcctc actctccgcc tccgtggggg accgcgtgac cattacctgt 480

cgggcatcac agtccatcag ctcatacctg aactggtatc agcagaagcc ggggaaggcc 540

ccgaaactcc tgatctacgc cgcctcctcc ctgcaatccg gcgtgccctc gaggttctcc 600

ggctccggct cgggaaccga tttcactctg acaattagca gcctgcagcc tgaggatttc 660

gctacctact actgccagca gtcctactcg actccgctga ctttcggcgg gggaaccaag 720

gtcgacatca ag 732

<210> 262

<211> 250

<212> PRT

<213> human

<400> 262

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Gly Ser Leu Pro Asp Tyr Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln

130 135 140

Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser

145 150 155 160

Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu

165 170 175

Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr

180 185 190

Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

195 200 205

Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu

210 215 220

Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu Gln Thr Leu Ile Thr

225 230 235 240

Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

245 250

<210> 263

<211> 251

<212> PRT

<213> human

<400> 263

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Arg Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Tyr Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Ser Pro Ser Gly Gly Ser Pro Thr Tyr Ala Gln Arg Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Leu Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Ser Arg Leu Arg Gly Asn Arg Leu Gly Leu Gln Ser Ser

100 105 110

Ile Phe Asp His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Asp Ile Arg Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Val Gly

145 150 155 160

Asp Arg Val Thr Ile Pro Cys Gln Ala Ser Gln Asp Ile Asn Asn His

165 170 175

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Gln Leu Leu Ile

180 185 190

Tyr Asp Thr Ser Asn Leu Glu Ile Gly Val Pro Ser Arg Phe Ser Gly

195 200 205

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

210 215 220

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Glu Asn Leu Pro Leu

225 230 235 240

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 264

<211> 250

<212> PRT

<213> human

<400> 264

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Thr Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Arg Ala Ser Asp Ser Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Ser Asp Tyr Asp Tyr Tyr Phe Asp Phe Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln

130 135 140

Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser

145 150 155 160

Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu

165 170 175

Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr

180 185 190

Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

195 200 205

Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu

210 215 220

Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu Gln Thr Pro Phe Thr

225 230 235 240

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

245 250

<210> 265

<211> 240

<212> PRT

<213> human

<400> 265

Gln Val Gln Leu Val Gln Ser Gly Gly Asp Leu Ala Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Pro Asp Gly Gly Gln Lys Tyr Tyr Gly Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Pro Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Val Arg His Phe Asn Ala Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu

130 135 140

Ser Ala Tyr Val Gly Gly Arg Val Thr Ile Thr Cys Gln Ala Ser Gln

145 150 155 160

Gly Ile Ser Gln Phe Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala

165 170 175

Pro Lys Leu Leu Ile Ser Asp Ala Ser Asn Leu Glu Pro Gly Val Pro

180 185 190

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile

195 200 205

Thr Asn Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr

210 215 220

Asp Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

225 230 235 240

<210> 266

<211> 248

<212> PRT

<213> human

<400> 266

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Lys

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Phe

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Tyr Asp Gly Ser Asn Ala Phe Tyr Ala Asp Ser Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asp Ser Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Ala Gly Asp Gly Gly Tyr Asp Val Phe Asp Ser Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser

130 135 140

Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys

145 150 155 160

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

165 170 175

Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu

180 185 190

Gly Ser Asn Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly

195 200 205

Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp

210 215 220

Val Gly Val Tyr Tyr Cys Met Gln Ala Leu Gln Thr Pro Thr Phe Gly

225 230 235 240

Pro Gly Thr Lys Val Asp Ile Lys

245

<210> 267

<211> 245

<212> PRT

<213> human

<400> 267

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Glu Thr Asp Tyr Tyr Gly Ser Gly Thr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln

130 135 140

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Ser

145 150 155 160

Cys Arg Ala Ser Gln Gly Ile Gly Ile Tyr Leu Ala Trp Tyr Gln Gln

165 170 175

Arg Ser Gly Lys Pro Pro Gln Leu Leu Ile His Gly Ala Ser Thr Leu

180 185 190

Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

195 200 205

Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Ser Tyr

210 215 220

Trp Cys Gln Gln Ser Asn Asn Phe Pro Pro Thr Phe Gly Gln Gly Thr

225 230 235 240

Lys Val Glu Ile Lys

245

<210> 268

<211> 244

<212> PRT

<213> human

<400> 268

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Gly Pro Ser Ser Trp Gly Glu Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Arg Leu Thr Gln Ser

130 135 140

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

145 150 155 160

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln

180 185 190

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

210 215 220

Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys

225 230 235 240

Val Asp Ile Lys

<210> 269

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 269

Ser Tyr Trp Ile Gly

1 5

<210> 270

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 270

Asn Tyr Tyr Val His

1 5

<210> 271

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 271

Ser Tyr Ala Met Ser

1 5

<210> 272

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 272

Ser Tyr Trp Ile Gly

1 5

<210> 273

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 273

Asp Tyr Ala Met His

1 5

<210> 274

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 274

Ile Phe Ala Met His

1 5

<210> 275

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 275

Ser Tyr Ala Met Ser

1 5

<210> 276

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 276

Asp Phe Phe Ile His

1 5

<210> 277

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 277

Asn Tyr Trp Ile Gly

1 5

<210> 278

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 278

Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 279

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 279

Ile Ile Ser Pro Ser Gly Gly Ser Pro Thr Tyr Ala Gln Arg Leu Gln

1 5 10 15

Gly

<210> 280

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 280

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 281

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 281

Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 282

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 282

Val Ile Trp Pro Asp Gly Gly Gln Lys Tyr Tyr Gly Asp Ser Val Lys

1 5 10 15

Gly

<210> 283

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 283

Thr Ile Ser Tyr Asp Gly Ser Asn Ala Phe Tyr Ala Asp Ser Val Glu

1 5 10 15

Gly

<210> 284

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 284

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 285

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 285

Trp Ile Asn Pro Asn Ser Gly Val Thr Lys Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 286

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 286

Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 287

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 287

Leu Gly Gly Ser Leu Pro Asp Tyr Gly Met Asp Val

1 5 10

<210> 288

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 288

Glu Ser Arg Leu Arg Gly Asn Arg Leu Gly Leu Gln Ser Ser Ile Phe

1 5 10 15

Asp His

<210> 289

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 289

Glu Asp Thr Ile Arg Gly Pro Asn Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 290

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 290

Gly Gly Tyr Ser Asp Tyr Asp Tyr Tyr Phe Asp Phe

1 5 10

<210> 291

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 291

His Phe Asn Ala Trp Asp Tyr

1 5

<210> 292

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 292

Ala Gly Asp Gly Gly Tyr Asp Val Phe Asp Ser

1 5 10

<210> 293

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 293

Glu Thr Asp Tyr Tyr Gly Ser Gly Thr Phe Asp Tyr

1 5 10

<210> 294

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 294

Trp Tyr Ser Ser Gly Trp Tyr Gly Ile Ala Asn Ile

1 5 10

<210> 295

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 295

His Gly Pro Ser Ser Trp Gly Glu Phe Asp Tyr

1 5 10

<210> 296

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 296

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 297

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 297

Gln Ala Ser Gln Asp Ile Asn Asn His Leu Asn

1 5 10

<210> 298

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 298

Arg Ala Ser Gln Asp Ile Asp Thr Trp Leu Ala

1 5 10

<210> 299

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 299

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 300

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 300

Gln Ala Ser Gln Gly Ile Ser Gln Phe Leu Asn

1 5 10

<210> 301

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 301

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 302

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 302

Arg Ala Ser Gln Gly Ile Gly Ile Tyr Leu Ala

1 5 10

<210> 303

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 303

Gln Ala Ser His Asp Ile Ser Asn Tyr Leu His

1 5 10

<210> 304

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 304

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 305

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 305

Leu Gly Ser Asn Arg Ala Ser

1 5

<210> 306

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 306

Asp Thr Ser Asn Leu Glu Ile

1 5

<210> 307

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 307

Ala Ala Ser Asn Leu Gln Gly

1 5

<210> 308

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 308

Leu Gly Ser Asn Arg Ala Ser

1 5

<210> 309

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 309

Asp Ala Ser Asn Leu Glu Pro

1 5

<210> 310

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 310

Leu Gly Ser Asn Arg Ala Ser

1 5

<210> 311

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 311

Gly Ala Ser Thr Leu Gln Ser

1 5

<210> 312

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 312

Asp Ala Ser Asn Leu Glu Thr

1 5

<210> 313

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 313

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 314

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 314

Met Gln Ala Leu Gln Thr Leu Ile Thr

1 5

<210> 315

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 315

Gln Gln Tyr Glu Asn Leu Pro Leu Thr

1 5

<210> 316

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 316

Gln Gln Ala Ser Ile Phe Pro Pro Thr

1 5

<210> 317

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 317

Met Gln Ala Leu Gln Thr Pro Phe Thr

1 5

<210> 318

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 318

Gln Gln Tyr Asp Asp Leu Pro Leu Thr

1 5

<210> 319

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 319

Met Gln Ala Leu Gln Thr Pro Thr

1 5

<210> 320

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 320

Gln Gln Ser Asn Asn Phe Pro Pro Thr

1 5

<210> 321

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 321

Gln Gln Ser Asp Asp Leu Pro His Thr

1 5

<210> 322

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 322

Gln Gln Ser Tyr Ser Thr Pro Leu Thr

1 5

<210> 323

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 323

Gly Tyr Ser Phe Thr Ser Tyr

1 5

<210> 324

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 324

Gly Tyr Ile Phe Thr Asn Tyr

1 5

<210> 325

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 325

Gly Phe Thr Phe Ser Ser Tyr

1 5

<210> 326

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 326

Gly Tyr Ser Phe Thr Ser Tyr

1 5

<210> 327

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 327

Gly Phe Thr Phe Asp Asp Tyr

1 5

<210> 328

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 328

Gly Phe Thr Phe Ser Ile Phe

1 5

<210> 329

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 329

Gly Phe Thr Phe Ser Ser Tyr

1 5

<210> 330

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 330

Gly Tyr Met Phe Thr Asp Phe

1 5

<210> 331

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 331

Gly Tyr Ser Phe Thr Asn Tyr

1 5

<210> 332

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 332

Tyr Pro Gly Asp Ser Asp

1 5

<210> 333

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 333

Ser Pro Ser Gly Gly Ser

1 5

<210> 334

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 334

Ser Gly Ser Gly Gly Ser

1 5

<210> 335

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 335

Tyr Pro Gly Asp Ser Asp

1 5

<210> 336

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 336

Trp Pro Asp Gly Gly Gln

1 5

<210> 337

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 337

Ser Tyr Asp Gly Ser Asn

1 5

<210> 338

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 338

Ser Gly Ser Gly Gly Ser

1 5

<210> 339

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 339

Asn Pro Asn Ser Gly Val

1 5

<210> 340

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 340

Tyr Pro Gly Asp Ser Asp

1 5

<210> 341

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 341

Leu Gly Gly Ser Leu Pro Asp Tyr Gly Met Asp Val

1 5 10

<210> 342

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 342

Glu Ser Arg Leu Arg Gly Asn Arg Leu Gly Leu Gln Ser Ser Ile Phe

1 5 10 15

Asp His

<210> 343

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 343

Glu Asp Thr Ile Arg Gly Pro Asn Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 344

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 344

Gly Gly Tyr Ser Asp Tyr Asp Tyr Tyr Phe Asp Phe

1 5 10

<210> 345

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 345

His Phe Asn Ala Trp Asp Tyr

1 5

<210> 346

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 346

Ala Gly Asp Gly Gly Tyr Asp Val Phe Asp Ser

1 5 10

<210> 347

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 347

Glu Thr Asp Tyr Tyr Gly Ser Gly Thr Phe Asp Tyr

1 5 10

<210> 348

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 348

Trp Tyr Ser Ser Gly Trp Tyr Gly Ile Ala Asn Ile

1 5 10

<210> 349

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 349

His Gly Pro Ser Ser Trp Gly Glu Phe Asp Tyr

1 5 10

<210> 350

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 350

Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr

1 5 10

<210> 351

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 351

Ser Gln Asp Ile Asn Asn His

1 5

<210> 352

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 352

Ser Gln Asp Ile Asp Thr Trp

1 5

<210> 353

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 353

Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr

1 5 10

<210> 354

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 354

Ser Gln Gly Ile Ser Gln Phe

1 5

<210> 355

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 355

Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr

1 5 10

<210> 356

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 356

Ser Gln Gly Ile Gly Ile Tyr

1 5

<210> 357

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 357

Ser His Asp Ile Ser Asn Tyr

1 5

<210> 358

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 358

Ser Gln Ser Ile Ser Ser Tyr

1 5

<210> 359

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 359

Leu Gly Ser

1

<210> 360

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 360

Asp Thr Ser

1

<210> 361

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 361

Ala Ala Ser

1

<210> 362

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 362

Leu Gly Ser

1

<210> 363

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 363

Asp Ala Ser

1

<210> 364

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 364

Leu Gly Ser

1

<210> 365

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 365

Gly Ala Ser

1

<210> 366

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 366

Asp Ala Ser

1

<210> 367

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 367

Ala Ala Ser

1

<210> 368

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 368

Ala Leu Gln Thr Leu Ile

1 5

<210> 369

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 369

Tyr Glu Asn Leu Pro Leu

1 5

<210> 370

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 370

Ala Ser Ile Phe Pro Pro

1 5

<210> 371

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 371

Ala Leu Gln Thr Pro Phe

1 5

<210> 372

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 372

Tyr Asp Asp Leu Pro Leu

1 5

<210> 373

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 373

Ala Leu Gln Thr Pro

1 5

<210> 374

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 374

Ser Asn Asn Phe Pro Pro

1 5

<210> 375

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 375

Ser Asp Asp Leu Pro His

1 5

<210> 376

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 376

Ser Tyr Ser Thr Pro Leu

1 5

<210> 377

<211> 150

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 377

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 150

<210> 378

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 378

Arg Gly Asp Ser

1

<210> 379

<211> 41

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 379

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 380

<211> 123

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 380

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 381

<211> 35

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polypeptide "

<400> 381

Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

1 5 10 15

Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp

20 25 30

Val Thr Leu

35

<210> 382

<211> 105

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 382

acaaaaaaga agtattcatc cagtgtgcac gaccctaacg gtgaatacat gttcatgaga 60

gcagtgaaca cagccaaaaa atccagactc acagatgtga cccta 105

<210> 383

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<400> 383

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 384

<211> 521

<212> DNA

<213> Unknown

<220>

<221> sources

<223 >/Annotation = "description of unknown sequence wild-type PGK promoter polynucleotide"

<400> 384

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtgtg 120

gggcggaggg cgtggcgggg aagggccggc gacgagagcc gcgcgggacg actcgtcggc 180

gataaccggt gtcgggtagc gccagccgcg cgacggtaac gagggaccgc gacaggcaga 240

cgctcccatg atcactctgc acgccgaagg caaatagtgc aggccgtgcg gcgcttggcg 300

ttccttggaa gggctgaatc cccgcctcgt ccttcgcagc ggccccccgg gtgttcccat 360

cgccgcttct aggcccactg cgacgcttgc ctgcacttct tacacgctct gggtcccagc 420

cgcggcgacg caaagggcct tggtgcgggt ctcgtcggcg cagggacgcg tttgggtccc 480

gacggaacct tttccgcgtt ggggttgggg caccataagc t 521

<210> 385

<211> 118

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 385

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtg 118

<210> 386

<211> 221

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 386

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtgtg 120

gggcggaggg cgtggcgggg aagggccggc gacgagagcc gcgcgggacg actcgtcggc 180

gataaccggt gtcgggtagc gccagccgcg cgacggtaac g 221

<210> 387

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 387

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtgtg 120

gggcggaggg cgtggcgggg aagggccggc gacgagagcc gcgcgggacg actcgtcggc 180

gataaccggt gtcgggtagc gccagccgcg cgacggtaac gagggaccgc gacaggcaga 240

cgctcccatg atcactctgc acgccgaagg caaatagtgc aggccgtgcg gcgcttggcg 300

ttccttggaa gggctgaatc cccg 324

<210> 388

<211> 422

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Polynucleotide "

<400> 388

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtgtg 120

gggcggaggg cgtggcgggg aagggccggc gacgagagcc gcgcgggacg actcgtcggc 180

gataaccggt gtcgggtagc gccagccgcg cgacggtaac gagggaccgc gacaggcaga 240

cgctcccatg atcactctgc acgccgaagg caaatagtgc aggccgtgcg gcgcttggcg 300

ttccttggaa gggctgaatc cccgcctcgt ccttcgcagc ggccccccgg gtgttcccat 360

cgccgcttct aggcccactg cgacgcttgc ctgcacttct tacacgctct gggtcccagc 420

cg 422

<210> 389

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<220>

<221> variants

<222> (1)..(3)

<223 >/replace = ""

<220>

<221> MISC_FEATURE

<222> (1)..(3)

<223 >/Note = "No preference given to variant residues in sequence with respect to description of variant position"

<400> 389

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 390

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<220>

<221> variants

<222> (1)..(3)

<223 >/replace = ""

<220>

<221> MISC_FEATURE

<222> (1)..(3)

<400> 390

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 391

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<220>

<221> variants

<222> (1)..(3)

<223 >/replace = ""

<220>

<221> MISC_FEATURE

<222> (1)..(3)

<400> 391

Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp

1 5 10 15

Val Glu Ser Asn Pro Gly Pro

20

<210> 392

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Annotation = "description of Artificial sequence-synthetic

Peptides "

<220>

<221> variants

<222> (1)..(3)

<223 >/replace = ""

<220>

<221> MISC_FEATURE

<222> (1)..(3)

<400> 392

Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala

1 5 10 15

Gly Asp Val Glu Ser Asn Pro Gly Pro

20 25

Claims

1. An isolated nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises a human CD33 binding domain, a transmembrane domain, and an intracellular signaling domain, and wherein the CD33 binding domain comprises a heavy chain complementary determining region 1(HC CDR1), a heavy chain complementary determining region 2(HC CDR2), and a heavy chain complementary determining region 3(HC CDR3) of any CD33 heavy chain binding domain amino acid sequence listed in table 2 or table 9.

2. The isolated nucleic acid molecule of claim 1, wherein the human CD33 binding domain further comprises a light chain complementary determining region 1(LC CDR1), a light chain complementary determining region 2(LC CDR2), and a light chain complementary determining region 3(LC CDR3) of any CD33 heavy chain binding domain amino acid sequence listed in table 2 or table 9.

3. The isolated nucleic acid molecule of claim 2, wherein the LC CDR1, LC CDR2, and LC CDR3 are the LC CDR sequences listed in table 11, 13, or 4.

4. The isolated nucleic acid molecule of claims 1-3, wherein the HC CDR1, HC CDR2, and HC CDR3 are the HC CDR sequences listed in Table 10, 12, or 3.

5. The isolated nucleic acid molecule of any of claims 1-4, which encodes a CAR comprising:

(i) the amino acid sequence of any light chain variable region listed in table 2 or table 9;

(ii) an amino acid sequence having at least one, two or three modifications, but no more than 30, 20 or 10 modifications of the amino acid sequence of any of the light chain variable regions provided in table 2 or 9; or

(iii) An amino acid sequence having 95-99% identity to the amino acid sequence of any of the light chain variable regions provided in table 2 or table 9.

6. The isolated nucleic acid molecule of any of claims 1-5, which encodes a CAR comprising:

(i) The amino acid sequence of any heavy chain variable region listed in table 2 or table 9;

(ii) an amino acid sequence having at least one, two or three modifications, but no more than 30, 20 or 10 modifications of the amino acid sequence of any of the heavy chain variable regions provided in table 2 or 9; or

(iii) An amino acid sequence having 95-99% identity to the amino acid sequence of any of the heavy chain variable regions provided in table 2 or table 9.

7. The isolated nucleic acid molecule of any of claims 1-6, which encodes a CAR comprising the amino acid sequence of any light chain variable region listed in Table 2 or Table 9 and the amino acid sequence of any heavy chain variable region listed Table 2 or Table 9.

8. The isolated nucleic acid molecule of any preceding claim, wherein the encoded CD33 binding domain comprises:

(i) an amino acid sequence selected from the group consisting of SEQ ID NOs 39-47, 57-65, 66-74 or 262-268;

(ii) an amino acid sequence having at least one, two or three modifications, but NO more than 30, 20 or 10 modifications to any of SEQ ID NOs 39-47, 57-65, 66-74 or 262-268; or

(iii) An amino acid sequence having 95-99% identity to any one of SEQ ID NOS 39-47, 57-65, 66-74 or 262-268.

9. The isolated nucleic acid molecule according to any of the preceding claims, wherein the CD33 binding domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NO 255-261 or a sequence having 95-99% identity thereto.

10. The isolated nucleic acid molecule of any preceding claim, wherein the encoded CAR comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of: the α, β or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154.

11. The isolated nucleic acid molecule of any preceding claim, wherein:

(i) the encoded transmembrane domain comprises the amino acid sequence of SEQ ID NO. 6, an amino acid sequence comprising at least one, two or three modifications, but NO more than 20, 10 or 5 modifications, of the amino acid sequence of SEQ ID NO. 6 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 6; or

(ii) The nucleic acid sequence encoding the transmembrane domain comprises the sequence of SEQ ID NO 17 or a sequence 95-99% identical thereto.

12. The isolated nucleic acid molecule of any preceding claim, wherein the encoded CD33 binding domain is connected to the transmembrane domain by a hinge region.

13. The nucleic acid molecule of claim 12, wherein:

(i) the encoded hinge region comprises the amino acid sequence of SEQ ID NO 2 or a sequence having 95-99% identity thereto; or

(ii) The nucleic acid sequence encoding the hinge region comprises the nucleotide sequence of SEQ ID NO 13 or a sequence having 95-99% identity thereto.

14. The isolated nucleic acid molecule of any preceding claim, wherein the encoded co-stimulatory domain is a functional signal domain obtained from a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signal molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1(CD11a/CD a), 4-1BB (CD137), B a-H a, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHT TR), KIRDS a, SLAMF a, NKp a (KLRF a), NKp a, CD a alpha, CD a beta, IL2 a gamma, ITIL 7 alpha, IT3672, VLITGA 72, VLITGA a, GAITGB 72, GAITGA a, GAITGB 11-a, GAITGA a, GAITGB 11, GAITGA a, GAITGB a, GAIT11, CD a, GAITGA a, GAITGB 72, GAITGA a, CD a, GAITGB 11-a, GAITGB 11, GAITGA a, GAITGB a, CD a, GAITGA a, GAITGB 11-a, GAITCD a, GAITGA a, CD a, GAITGB 11-a, CD a, GAITGB 11, SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds to CD 83.

15. The isolated nucleic acid molecule according to claim 14, wherein the encoded co-stimulatory domain comprises the amino acid sequence of SEQ ID No. 7, or an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID No. 7 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID No. 7.

16. The isolated nucleic acid molecule of claim 14, wherein the nucleic acid sequence encoding the co-stimulatory domain comprises the nucleotide sequence of SEQ ID No. 18 or a sequence 95-99% identical thereto.

17. The isolated nucleic acid molecule of any preceding claim, wherein the encoded intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 ζ.

18. The isolated nucleic acid molecule of any preceding claim, wherein the encoded intracellular signaling domain comprises the amino acid sequence of SEQ ID No. 7 and/or the sequence of SEQ ID No. 9 or SEQ ID No. 10; having the amino acid sequence of SEQ ID NO. 7 and/or at least one, two or three modifications, but not more than 20, 10 or 5 modifications, of the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10 or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO. 7 and/or the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10.

19. The isolated nucleic acid molecule of any preceding claim, wherein the encoded intracellular signaling domain comprises the sequence of SEQ ID NO 7 and the sequence of SEQ ID NO 9 or SEQ ID NO 10, wherein the sequences comprising the intracellular signaling domain are expressed in frame and as a single polypeptide chain.

20. The isolated nucleic acid molecule of any preceding claim, wherein the nucleic acid sequence encoding the intracellular signaling domain comprises the sequence of SEQ ID NO. 18 or a sequence 95-99% identical thereto, and/or the sequence of SEQ ID NO. 20 or SEQ ID NO. 21 or a sequence 95-99% identical thereto.

21. The isolated nucleic acid molecule of any preceding claim, further comprising a leader sequence encoding the amino acid sequence of SEQ ID NO 1.

22. The isolated nucleic acid molecule of any preceding claim, which encodes a CAR comprising:

(i) any one of SEQ ID NO 48-56;

(ii) an amino acid sequence having at least one, two or three modifications, but NO more than 30, 20 or 10 modifications to any of SEQ ID NOs 48-56; or

(iii) An amino acid sequence having 95-99% identity to any one of SEQ ID NOs 48-56.

23. The isolated nucleic acid molecule of any preceding claim, comprising the nucleotide sequence of any one of SEQ ID NOs 75-83 or a nucleotide sequence having 95-99% identity to any one of SEQ ID NOs 75-83.

24. An isolated polypeptide molecule encoded by the nucleic acid molecule of any one of claims 1-23.

25. An isolated Chimeric Antigen Receptor (CAR) polypeptide, wherein the CAR comprises an antibody or antibody fragment comprising a human CD33 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain, and wherein the CD33 binding domain comprises heavy chain complementarity determining region 1(HC CDR1), heavy chain complementarity determining region 2(HC CDR2), and heavy chain complementarity determining region 3(HC CDR3) of any CD33 heavy chain binding domain amino acid sequence listed in table 2 or table 9.

26. The isolated CAR polypeptide of claim 25, wherein the human CD33 binding domain further comprises a light chain complementary determining region 1(LC CDR1), a light chain complementary determining region 2(LC CDR2), and a light chain complementary determining region 3(LC CDR3) of any CD33 heavy chain binding domain amino acid sequence listed in table 2 or table 9.

27. The isolated CAR polypeptide of claim 26, wherein the LC CDR1, LC CDR2, and LC CDR3 are the LC CDR sequences listed in table 11, 13, or 4.

28. The isolated CAR polypeptide of claims 25-27, wherein the HC CDR1, HC CDR2, and HC CDR3 are the HC CDR sequences listed in table 10, 12, or 3.

29. The isolated CAR polypeptide of any of claims 25-28, comprising:

30. The isolated CAR polypeptide of any of claims 25-29, comprising:

31. The isolated CAR polypeptide of any of claims 25-30, comprising the amino acid sequence of any light chain variable region listed in table 2 or 9 and the amino acid sequence of any heavy chain variable region listed table 2 or 9.

32. The isolated CAR polypeptide of any of claims 25-31, comprising:

(i) any one amino acid sequence selected from the group consisting of SEQ ID NOs 39-47, 57-65, 66-74, and 262-268;

33. The isolated CAR polypeptide of any of claims 25-32, wherein the transmembrane domain comprises a transmembrane domain from a protein selected from the group consisting of: the α, β or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154.

34. The isolated CAR polypeptide of any of claims 25-33, wherein:

(i) the transmembrane domain comprises the amino acid sequence of SEQ ID NO 6,

(ii) the amino acid sequence comprises at least one, two or three modifications, but not more than 20, 10 or 5 modifications, of the amino acid sequence of SEQ ID NO 6, or

(iii) A sequence having 95-99% identity to the amino acid sequence of SEQ ID NO 6.

35. The isolated CAR polypeptide of any of claims 25-34, wherein the CD33 binding domain is connected to the transmembrane domain by a hinge region.

36. The isolated CAR polypeptide of claim 35, wherein the hinge region comprises SEQ ID No. 2 or a sequence 95-99% identical thereto.

37. The isolated CAR polypeptide of any of claims 25-36, wherein co-stimulatory domain is a functional signal domain obtained from a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signal molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1(CD11a/CD a), 4-1BB (CD137), B a-H a, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHT TR), KIRDS a, SLAMF a, NKp a (KLRF a), NKp a, CD a alpha, CD a beta, IL2 a gamma, ITIL 7 alpha, IT3672, VLITGA 72, VLITGA a, GAITGB 72, GAITGA a, GAITGB 11-a, GAITGA a, GAITGB 11, GAITGA a, GAITGB a, GAIT11, CD a, GAITGA a, GAITGB 72, GAITGA a, CD a, GAITGB 11-a, GAITGB 11, GAITGA a, GAITGB a, CD a, GAITGA a, GAITGB 11-a, GAITCD a, GAITGA a, CD a, GAITGB 11-a, CD a, GAITGB 11, SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds to CD 83.

38. The isolated CAR polypeptide of any of claims 25-37, wherein the co-stimulatory domain comprises the amino acid sequence of SEQ ID No. 7, or an amino acid sequence having at least one, two or three modifications, but NO more than 20, 10 or 5 modifications, of the amino acid sequence of SEQ ID No. 7, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID No. 7.

39. The isolated CAR polypeptide of any of claims 25-37, wherein intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 ζ.

40. The isolated CAR polypeptide of any of claims 25-39, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID No. 7 and/or the sequence of SEQ ID No. 9 or SEQ ID No. 10; an amino acid sequence having at least one, two or three modifications, but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO 7 and/or the amino acid sequence of SEQ ID NO 9 or SEQ ID NO 10; or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 7 and/or the amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 10.

41. The isolated CAR polypeptide of any of claims 25-40, wherein the intracellular signaling domain comprises the sequence of SEQ ID No. 7 and the sequence of SEQ ID No. 9 or SEQ ID No. 10, wherein the sequences comprising the intracellular signaling domain are expressed in frame and as a single polypeptide chain.

42. The isolated CAR polypeptide of any of claims 25-41, further comprising a leader sequence comprising the amino acid sequence of SEQ ID NO 1.

43. The isolated CAR polypeptide of any of claims 25-42, comprising:

(i) any one of SEQ ID NO 48-56;

44. A vector comprising a nucleic acid molecule encoding a CAR or CD33 binding domain according to any preceding claim, wherein the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector or a retroviral vector.

45. The vector of claim 44, further comprising an EF-1 promoter comprising the sequence of SEQ ID NO 11.

46. A cell, e.g., an immune effector cell, comprising the nucleic acid of any one of claims 1-23, the CAR polypeptide of any one of claims 24-43, or the vector of claim 44 or 45.

47. A method of making a cell, such as an immune effector cell, comprising transducing an immune effector cell with the vector of claim 44 or 45.

48. A method of producing a population of RNA-engineered cells, comprising introducing into a cell an in vitro transcribed RNA or a synthetic RNA, wherein the RNA comprises a nucleic acid encoding the CAR polypeptide of any preceding claim.

49. A method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell, e.g., a population of immune effector cells, comprising the CAR nucleic acid of any one of claims 1-23 or the CAR polypeptide of any one of claims 24-43.

50. The method of claim 49, wherein the cells are autologous T cells or allogeneic T cells.

51. A method of treating a mammal having a disease associated with expression of CD33, comprising administering to the mammal an effective amount of a cell, e.g., a population of immune effector cells, comprising the CAR nucleic acid of any one of claims 1-23 or the CAR polypeptide of any one of claims 24-43.

52. The method of claim 51, wherein the disease associated with expression of CD33 is:

(i) a cancer or malignancy or a precancerous condition selected from one or more of myelodysplasia, myelodysplastic syndrome, or a pre-leukemic stage, or

(ii) Non-cancer related indications associated with expression of CD 33.

53. The method of claim 51 or 52, wherein the disease is a hematological cancer.

54. The method of any one of claims 51-53, wherein the disease is acute leukemia selected from Acute Myeloid Leukemia (AML); myelodysplastic syndrome; myeloproliferative tumors; chronic Myeloid Leukemia (CML); or a blast plasma cell-like dendritic cell tumor or a combination thereof.

55. The method of any one of claims 51-54, wherein cells, e.g., a population of immune effector cells, are administered in combination with one or more of the following:

(i) an agent that increases the efficacy of a cell comprising a CAR nucleic acid or CAR polypeptide;

(ii) an agent that mitigates one or more side effects associated with administration of a cell comprising a CAR nucleic acid or CAR polypeptide; or

(iii) An agent for treating a disease associated with CD 33.

56. The isolated nucleic acid molecule of any one of claims 1-23, the isolated CAR polypeptide molecule of any one of claims 24-43, the vector of claim 44 or 45, or the cell of claim 46, for use as a medicament.

57. The isolated nucleic acid molecule of any one of claims 1-23, the isolated CAR polypeptide molecule of any one of claims 24-43, the vector of claim 44 or 45, or the cell of claim 46, for use in treating a disease associated with expression of CD 33.

58. The cell of claim 49, e.g., a population of immune effector cells, further expressing an inhibitory molecule comprising a first polypeptide comprising at least a portion of an inhibitory molecule associated with a second polypeptide comprising a forward signal from an intracellular signaling domain.

59. The cell of claim 58, wherein the inhibitory molecule comprises a first polypeptide comprising at least a portion of PD1 and a second polypeptide comprising a costimulatory domain and a primary signaling domain.

60. The method of claim 55, wherein the agent is an mTOR inhibitor and the subject is administered an immunopotentiating low dose of an mTOR inhibitor, e.g., RAD001 or rapamycin.

61. The method of claim 60, wherein the mTOR inhibitor is administered for a sufficient amount of time to decrease the proportion of PD-1 positive T cells, increase the proportion of PD-1 negative T cells, or increase the ratio of PD-1 negative T cells/PD-1 positive T cells in the peripheral blood of the subject or in a T cell preparation isolated from the subject.

62. A method of conditioning a subject prior to cell transplantation comprising administering to the subject an effective amount of a cell comprising the CAR nucleic acid molecule of any one of claims 1-23 or the CAR polypeptide of any one of claims 24-43.

63. The method of claim 62, wherein cell transplantation is stem cell transplantation, such as hematopoietic stem cell transplantation, or bone marrow transplantation.

64. The method of claim 62 or 63, wherein conditioning the subject prior to cell transplantation comprises reducing the number of CD33 expressing cells, such as CD33 expressing normal cells or CD33 expressing cancer cells, in the subject.